Home

Procise® Protein Sequencing System

Contents

1. Step Action 1 Select the Function view from the View pop up menu 2 Highlight the function from the function list and enter the desired global time in the global value field 3 Pull down the File menu from the upper menu bar and select Save Functions Activating Global To activate the global time Time Step Action 1 Use the View menu to select the Cycles amp Procedures view 2 Use the Cycle menu to select the type of cycle or procedure in which the global time will be activated Then select the particular cycle to be modified Highlight the step where the global time will be activated Check the global box to activate the global time An X in the global box indicates that the global time is active Pull down the File menu from the upper menu bar and select Save Cycle Procedure Creating Functions Cycles and Methods 4 3 Creating Cycles Modifying Existing To make a copy of the Procise standard cartridge cycle Cartridge Cycles Step Action 1 Use the View menu to select the Cycles and Procedures View Use the upper screen menu to select Cartridge Cycle for cycle type Use the lower screen menu to select the specific cycle to be copied 2 3 4 Pull down the File menu from the upper menu bar and select Save Cycle Procedures As Type the new cycle name and click the OK button Editing the Copied To edi
2. Premix Buffer Concentrate CHEMICAL HAZARD Premix Buffer Concentrate causes burns to the eyes skin and respiratory tract It is a combustible liquid and vapor Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves R1 5 phenylisothiocyanate in n heptane CHEMICAL HAZARD R1 5 phenylisothiocyanate in n heptane causes burns to the eyes skin and respiratory tract It is a flammable liquid and vapor Exposure may cause an allergic skin reaction and central nervous system effects such as drowsiness dizziness headache etc and irregular heartbeats Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves R2B n methylpiperidine in methanol and water CHEMICAL HAZARD R2B n methylpiperidine in methanol and water is a flammable liquid and vapor Exposure may cause eye skin and respiratory tract irritation and central nervous system depression and blindness Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves R3 trifluoroacetic acid CHEMICAL HAZARD R3 trifluoroacetic acid causes severe burns to the eyes skin and respiratory tract Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves R4 25 trifluoroacetic acid in water CHEMICAL HAZA
3. Eastern Asia China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10 64106608 86 10 64106617 Hong Kong 852 2756 6928 852 2756 6968 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 758 8268 60 3 754 9043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 22358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 35 75 0 43 0 1 867 35 75 11 Belgium 32 0 2 712 5555 32 0 2 712 5516 Czech Republic and Slovakia Praha 420 2 61 222 164 420 2 61 222 168 Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 101 0 49 0 6150 101 101 Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 231206 05 47 23 12 05 75 Poland Lithuania Latvia and Estonia 48 22 866 40 10 48 22 866 40 20 Warszawa Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Russia Moskva 7 095 935 8888 7 095 564 8787 South East Europe Zagreb Croatia 385 1 34 91 927 385 1 34 91 840 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 1206 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41
4. Topic See Page Components of a Run 3 2 Valves 3 3 Functions 3 6 Cycles 3 9 Methods 3 11 Valves Functions Cycles and Methods 3 1 Components of a Run Table of Definitions of the components that constitute a run are described below Each Components component is discussed in this chapter Run Component Definition Valve A valve is a mechanical device which opens to provide a flow path for the transfer of gas solvent or reagent The Procise Protein Sequencer has three types of valves that perform one of the following functions Deliver liquids vapors or gas Deliver only gas Vent the chemical bottles Function Functions activate a valve or a set of valves to deliver a chemical activate or deactivate a relay define or increment a setpoint or tell the pump to start or stop Customized functions can be created by the user Step A function once incorporated into a cycle becomes a step of that cycle This function may either have a fixed or a global time associated with it Cycle A cycle is a series of steps in an order specified by the standard chemistry or by the user that accomplishes a specific chemical process in the reaction cartridge and or the conversion flask Standard cycles those supplied by Applied Biosystems are permanently stored in memory The user can also create new cycles or edit existing cycles Method A method consists of the cycles repetitio
5. O N Oa R 0 PM ND N O O N O G sa 0 MN O B 30 Cycle Method and Gradient Listings Function 258 235 218 215 173 213 218 228 213 301 236 301 218 153 213 218 173 218 301 213 237 Function Name End Function Name Begin Load Position Wait Prepare Pump Wait Inject Pos Collect Data Start Gradient Wait Wait End Function Name Begin Set as Residue Cycle Flush Large Loop Flask Empty Flask Load S4 Flask Ig loop Dry Flask Flush Large Loop Flask Ready to Receive Dry Flask Pause Pre Conversion Dry Pause Flush Large Loop Flask Load R4 Flask Ig loop Dry Flask Flush Large Loop Flask Load S4 Flask Ig loop Flush Large Loop Flask Pause Dry Flask Post Conversion Dry Time sec 0 Time sec 0 1 30 1 480 690 690 Time sec 0 0 10 20 15 10 10 1 120 10 20 10 10 15 10 450 Global Time N Global Time Z Z ZZ Z ZZ Z Z Z Global Time N KZ ZZ ZZ Z ZZZKxX zz Z ZZ Zz Z Z Z Global Step Function Function Name Time sec Time 22 259 End 0 N Cycle Method and Gradient Listings B 31 Method List B 32 Methods Table Method Name Filter Precycle Fast Precycle Pulsed liquid Gas phase PL PVDF Protein GP PVDF Protein PL PVDF Peptide Cycle Method and Gradient Listings Default Default 1 Default Default 1 2 3 Default Default Defau
6. 10 20 0 Global Time N Z Z Z ZZ ZZ Cleanup Procedure List Delivery Line Total run time 16 25 Backflush Step AN Oa R 0 PM NN N M M NNN D a aaa 1 0 N O 0d WBN O DAN DA BR ON O Function 258 7 9 17 19 27 29 39 47 49 59 69 77 79 89 97 99 206 154 156 164 166 174 176 184 186 196 259 Cartridge Valve Total run time 12 56 Block Wash S1 Step ROD Function 258 103 104 101 Function Name Begin Vent R1 Backflush R1 Vent R2g Backflush R2g Vent R3 Backflush R3 Backflush R3g Vent S1 Backflush S1 Backflush S2 Backflush S3 Vent X1 Backflush X1 Backflush X1g Vent X3 Cart Backflush X3 Cart Backflush X3 Flask Vent R4 Backflush R4 Vent R5 Backflush R5 Vent S4 Backflush S4 Vent X2 Backflush X2 Backflush X2g End Function Name Begin Wash Reagent Block S1 Wash Solvent Block S1 Wash Input Block S1 Time sec 0 5 60 10 60 5 60 60 5 60 60 60 5 60 60 5 60 60 5 60 5 30 5 60 5 60 60 0 Time sec 0 180 180 180 Procedure Listings C 17 Global Time Z Z Z Z Z Z z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Global Time N N N N C 18 Procedure Listings Step O N O Aa 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Function 123 102 41 42 41 42 41 42 41 42 41 148 41 148 42 147
7. 63 131 63 131 63 131 259 Cart Precycle Total run time 34 20 Step O N o asa 0 PM M PO N N N N N N a H A A BH da S CE ND OF 0 N O O O N N DOA ON O B 6 Cycle Method and Gradient Listings Function 258 143 135 11 140 6 131 140 135 11 132 140 6 131 140 135 11 146 140 143 135 136 132 63 61 148 51 Function Name Cartridge Wait Dry Cart top Del S3 Cart sensor Dry Cart top Del S3 Cart Dry Cart top sensor Del S3 Cart sensor Dry Cart top End Function Name Begin Wash Cart Reagent Block Flush Cart Reagent Block Del R2g Cart top Flush Large Loop Cart Load R1 Cart Ig loop Dry Cart top Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Dry Cart bottom Flush Large Loop Cart Load R1 Cart Ig loop Dry Cart top Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Wash Large Loop Cart Flush Large Loop Cart Wash Cart Reagent Block Flush Cart Reagent Block Flush Cart Solvent Block Dry Cart bottom Del S3 Cart sensor Del S3 Cart top Cartridge Wait Del S2 Cart top Time sec 5 30 20 30 20 30 20 60 0 Time sec 0 10 30 60 10 20 30 5 5 170 30 5 20 30 5 5 170 10 20 15 30 30 60 20 10 5 10 Global Time N ZIZ Z Z Z Z ZZ Global Time N Z Z ZZ Z ZZ ZZ ZZ Zz ZZ LL Zi 2 LL EL ze zz z Step 28 29 3
8. CHEMICAL HAZARD Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Argon CHEMICAL HAZARD Argon is a nonflammable high pressure gas Released argon gas reduces the oxygen available for breathing Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves B2 12 isopropanol and acetonitrile CHEMICAL HAZARD B2 12 isopropanol and acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation Prolonged or repeated contact may dry skin Exposure may cause central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Biobrene Plus CHEMICAL HAZARD Biobrene Plus may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Dithiothreitol DTT I M Willi CHEMICAL HAZARD Dithiothreitol DTT may cause eye skin and respiratory tract irritation central In R4A R5 nervous system depression and damage to the kidneys Please read the MSDS
9. Every cycle needs a Begin step and an End step Creating Functions Cycles and Methods 4 5 Creating New Cycles To select User Defined Cycle 1 Action Use the View menu to select Cycles and Procedures View Use the upper screen menu to select the cycle type Using a User Defined Cycle Step 1 2 3 Use the lower screen menu to select the User Defined Cycle 1 Editing the New To edit the new cycle Cycle Step Action 1 To delete a row highlight the row to be deleted and click the Delete Row button 2 To insert a row select the function to be inserted from the function list on the left side of the screen The function can be selected by either using the scroll down button or typing the function number at the top corner of the function list a Highlight the row immediately before the intended insertion point b To enter the function run time click on the global box to turn the global time off Type the function time in seconds in the value box Saving the New To save the new cycle Cycle Step Action 1 Pull down the File menu from the upper menu bar 2 Select Save Cycle Procedures As 3 Type the new cycle name and click the OK button IMPORTANT The maximum number of steps allowed per cycle is 100 A cartridge must have Ready to Transfer and Transfer Complete steps to receive sample from the cartridge Every
10. Flush Large Loop Cart Flush Input Block Time sec 10 45 5 15 10 45 0 60 0 Time sec 0 5 30 10 20 30 5 5 170 30 5 20 30 5 5 170 30 5 20 30 5 5 170 10 10 15 20 Global Time N ZIZ Z Z Z Z ZZ Global Time N Z Z ZZ Z ZZ ZZ Z ZZ ZZzZz Z Z Z ZZ Z ZZ ZZ Z Global Step Function Function Name Time sec Time 28 136 Flush Cart Solvent Block 30 N 29 132 Dry Cart bottom 60 N 30 142 Set Cart Temperature 53 N 31 63 Del S3 Cart sensor 15 N 32 148 Cartridge Wait 10 N 33 61 Del S3 Cart top 10 N 34 148 Cartridge Wait 10 N 35 51 Del S2 Cart top 5 N 36 148 Cartridge Wait 5 N 37 51 Del S2 Cart top 5 N 38 148 Cartridge Wait 5 N 39 131 Dry Cart top 30 N 40 53 Del S2 Cart sensor 15 N 41 148 Cartridge Wait 5 N 42 51 Del S2 Cart top 5 N 43 148 Cartridge Wait 5 N 44 51 Del S2 Cart top 5 N 45 148 Cartridge Wait 5 N 46 61 Del S3 Cart top 10 N 47 148 Cartridge Wait 10 N 48 131 Dry Cart top 60 N 49 137 Flush Input Block 30 N 50 140 Flush Large Loop Cart 10 N 51 26 Load R3 Cart Ig loop 50 N 52 30 Transfer R3 Cart gas 5 N 53 136 Flush Cart Solvent Block 10 N 54 140 Flush Large Loop Cart 10 N 55 138 Flush Output Block 10 N 56 144 Wash Cart Solvent Block 10 N 57 143 Wash Cart Reagent Block 15 N 58 146 Wash Large Loop Cart 10 N 59 107 Wash Output Block S2 15 N 60 137 Flush Input Block 10 N 61 136 Flush Cart Solvent Block 10 N 62 111 Wash Input Block
11. Global Step Function Function Name Time sec Time 13 131 Dry Cart top 30 N 14 140 Flush Large Loop Cart 5 N 15 135 Flush Cart Reagent Block 5 N 16 11 Del R2g Cart top 170 N 17 132 Dry Cart bottom 30 N 18 140 Flush Large Loop Cart 5 N 19 6 Load R1 Cart Ig loop 20 N 20 131 Dry Cart top 30 N 21 140 Flush Large Loop Cart 5 N 22 135 Flush Cart Reagent Block 5 N 23 11 Del R2g Cart top 170 N 24 146 Wash Large Loop Cart 10 N 25 111 Wash Input Block S3 10 N 26 140 Flush Large Loop Cart 15 N 27 137 Flush Input Block 20 N 28 136 Flush Cart Solvent Block 30 N 29 132 Dry Cart bottom 60 N 30 142 Set Cart Temperature 48 N 31 63 Del S3 Cart sensor 15 N 32 148 Cartridge Wait 10 N 33 61 Del S3 Cart top 10 N 34 148 Cartridge Wait 10 N 35 51 Del S2 Cart top 5 N 36 148 Cartridge Wait 5 N 37 51 Del S2 Cart top 5 N 38 148 Cartridge Wait 5 N 39 131 Dry Cart top 30 N 40 53 Del S2 Cart sensor 15 N 41 148 Cartridge Wait 5 N 42 51 Del S2 Cart top 5 N 43 148 Cartridge Wait 5 N 44 51 Del S2 Cart top 5 N 45 148 Cartridge Wait 5 N 46 61 Del S3 Cart top 10 N 47 148 Cartridge Wait 10 N 48 131 Dry Cart top 60 N 49 137 Flush Input Block 30 N 50 140 Flush Large Loop Cart 10 N 51 26 Load R3 Cart Ig loop 50 N 52 30 Transfer R3 Cart gas 5 N 53 136 Flush Cart Solvent Block 10 N Cycle Method and Gradient Listings B 9 Step 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
12. Optimizing The Global Time Values 6 4 Chemical Optimization Post Conversion Dry steps in all flask cycles Note We recommend that you optimize these values whenever the flask and pickup line are cleaned or replaced The following procedures are provided here A procedure for setting up the protein sequencer A procedure for starting a run A procedure for continuing the run A procedure for calculating the optimized pre and post conve A procedure for verifying the optimized settings To set up the protein sequencer rsion dry step times Step Action 1 Install a reaction cartridge in the cartridge A position on the protein sequencer 2 Perform a leak test on cartridge A 3 Scroll to the Start Run dialog box E PROCISE Procise 1 0 File Edit Sequencer Help Start Run v Idle Stop ROT Pause Now Cartridge C File Name Cartridge 4 File Name roz Flask Opt GLC 12062 Cycles 30 El Cycles Method Run 151 x Pause later Flask Optimization c W Status Idle ldle Idle Status M Collect Data f Collect Data Sample 10 0 pmol O Stdiko o pmol pmol Sampl pmol f Colect Data std o ldle pmol pmol Startup None v Shutdown None Y Configure cartridge A as follows Parameter Cartridge A Filename Setting 1st Your choice 5 Flask Optimization Number of
13. Save Regulator Setpoint Vent R3 Backflush R3 Backflush R3g Pause for Bottle Change Set Reg Setpoint 10th psi Flush R3 Del R3 Waste Del R3g Waste Time sec 0 0 2 0 10 10 0 38 20 15 0 15 38 Time sec 0 0 3 0 10 60 20 0 15 15 60 20 Global Time Z Z ZIZ Z 2 2 2 NN LL LL Lie LL Z N SN ZZ Global Time N ZZ 2 zz Z Zz iz z z Step 13 14 15 16 Function 309 144 136 259 Bottle Change for Total run time 4 50 R3 Leak Step O N OOA ON 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Function 258 263 303 304 27 29 39 260 305 310 257 308 257 307 317 310 257 318 305 28 24 34 309 144 136 259 Function Name Restore Reg Setpoint Wash Cart Solvent Block Flush Cart Solvent Block End Function Name Begin Set for Bottle R3 Select Regulator Save Regulator Setpoint Vent R3 Backflush R3 Backflush R3g Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Set Reg Setpoint 10th psi Flush R3 Del R3 Waste Del R3g Waste Restore Reg Setpoint Wash Cart Solvent Block Flush Cart Solvent Block End Global Time sec Time 0 10 20 0 N N N N Global Time sec Time 0 0 3 0 10 60 20 0 38 20 30 0 15 38 0 5 30 0 15 15
14. TEA E ZZ ZZ Z ZZ Zz Z Zz Z ZZ zz Step 20 21 22 23 24 25 Function 8 4 135 143 135 259 R2 Leak Test Total run time 1 40 R3 Leak Test Step AN DU FWD 11 12 13 14 15 16 17 18 19 Function 258 303 304 305 310 257 308 257 307 317 310 257 318 17 310 307 309 18 259 Total run time 3 55 Step oa E 0 MN Function 258 303 304 27 29 305 Function Name Flush R1 Del R1 Waste Flush Cart Reagent Block Wash Cart Reagent Block Flush Cart Reagent Block End Function Name Begin Select Regulator Save Regulator Setpoint Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Vent R2g Set Tolerance 100th psi Compare Pressures 10th psi Restore Reg Setpoint Flush R2g End Function Name Begin Select Regulator Save Regulator Setpoint Vent R3 Backflush R3 Set Reg Setpoint 10th psi Time sec 5 10 10 10 30 0 Time sec 0 2 0 38 20 15 1 15 38 Time sec 0 3 0 10 20 38 Procedure Listings Global Time N Z Z Z Z Z Global Time Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Global Time N Z Z Z Z Z C 35 Step 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Function 310 257 308 257 307 317 310 257 318 27 310
15. odor R2B provides the basic environment necessary for PITC to react with the amino terminus of the sample In the standard cycles R2B is delivered to the filter cartridge between three R1 deliveries and argon drying steps IMPORTANT Oxidation Once coupling has occurred the PTC peptide or PTC protein is susceptible to oxidative de sulfurization Replacement of sulfur with oxygen inhibits cyclization and cleavage thus reducing yields For this reason all solvents and reagents are stored and maintained under argon and should be purged with argon whenever replaced Sequence Analysis Chemistry 2 3 Post Coupling Extraction Extracting with Solvents S2B and S3 Cleavage Diagram Cleaving with R3 ATZ Transfer Solvents are delivered through the filter to extract unwanted materials that could hinder later reactions or form byproducts which could interfere with the chromatographic analysis of PTH amino acids The solvents help remove the following PITC R1 byproducts generally thioureas and ureas Residual PITC to minimize additional thiourea formation Residual N methylpiperidine R2B to avoid salt formation during TFA R3 delivery Residual water for which BioBrene Plus has an affinity to minimize acid catalyzed hydrolysis during cleavage S2B ethyl acetate and S3 butyl chloride remove the majority of these unwanted materials The amounts of S2B and S3 delivered can be increased beyond that listed in the standard
16. 10 10 10 10 15 10 15 10 10 10 10 30 30 30 60 30 40 20 10 5 30 20 Global Time N Z EA Z Z ZZ ZZ Z Z Z ZZ ZZ Zz ZZ Z AN AS O Z ZZ Z Z ZZ Z Z Z Z Z Global Step Function Function Name Time sec Time 38 148 Cartridge Wait 5 N 39 51 Del S2 Cart top 5 N 40 148 Cartridge Wait 5 N 41 61 Del S3 Cart top 5 N 42 148 Cartridge Wait 5 N 43 61 Del S3 Cart top 5 N 44 148 Cartridge Wait 5 N 45 61 Del S3 Cart top 5 N 46 131 Dry Cart top 120 N 47 137 Flush Input Block 5 N 48 11 Del R2g Cart top 30 N 49 140 Flush Large Loop Cart 10 N 50 6 Load RI Cart Ig loop 20 N 51 131 Dry Cart top 30 N 52 140 Flush Large Loop Cart 5 N 53 135 Flush Cart Reagent Block 5 N 54 11 Del R2g Cart top 170 N 55 132 Dry Cart bottom 30 N 56 140 Flush Large Loop Cart 5 N 57 6 Load R1 Cart lg loop 20 N 58 131 Dry Cart top 30 N 59 140 Flush Large Loop Cart 5 N 60 135 Flush Cart Reagent Block 5 N 61 11 Del R2g Cart top 170 N 62 146 Wash Large Loop Cart 10 N 63 140 Flush Large Loop Cart 20 N 64 143 Wash Cart Reagent Block 15 N 65 135 Flush Cart Reagent Block 30 N 66 136 Flush Cart Solvent Block 30 N 67 132 Dry Cart bottom 60 N 68 63 Del S3 Cart sensor 20 N 69 131 Dry Cart top 30 N 70 63 Del S3 Cart sensor 20 N 71 131 Dry Cart top 30 N 72 53 Del S2 Cart sensor 20 N 73 51 Del S2 Cart top N 74 148 Cartridge Wait N 75 51 Del S2 Cart top 5 N 76 131 Dry Cart top 30 N 77 53 Del 82 C
17. 124 257 41 42 41 42 41 42 41 42 41 148 41 148 42 147 125 257 41 42 41 42 41 42 41 Function Name Select Cartridge A Wash Output Block S1 Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart top Del S1 Cart bottom Del S1 Cart top Cartridge Wait Del S1 Cart top Cartridge Wait Del S1 Cart bottom End Cartridge Select Select Cartridge B Wait Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart top Del S1 Cart bottom Del S1 Cart top Cartridge Wait Del S1 Cart top Cartridge Wait Del S1 Cart bottom End Cartridge Select Select Cartridge C Wait Del S1 Cart top Del S1 Cart bottom Del S1 Cart top Del S1 Cart bottom top bottom top bottom top bottom m A aa E top bottom top bottom top bottom n gem ml Jin IDR PERNE Del S1 Cart top Del S1 Cart bottom Del S1 Cart top Time sec 0 180 120 10 10 10 10 10 10 10 60 30 60 30 60 0 0 120 120 10 10 10 10 10 10 10 60 30 60 30 60 120 120 10 10 10 10 10 10 Global Time N Z Zz Z Ziz Z Zz ZZZ ZZ ZZ z Z LL 2 Zi ZZZ ZZZ Zz Z Z Z Zz z Z Step 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 Function 42 41 148 41 148 42 147 126
18. 148 131 34 31 143 135 144 136 131 142 127 141 63 148 121 141 53 148 121 141 63 148 121 128 131 61 148 131 259 Function Name Del S2 Cart sensor Cartridge Wait Del S2 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Del S3 Cart top Cartridge Wait Dry Cart top Del R3g Waste Del R3g Cart top Wash Cart Reagent Block Flush Cart Reagent Block Wash Cart Solvent Block Flush Cart Solvent Block Dry Cart top Set Cart Temperature Ready Transfer to Flask Flush Transfer Line Del S3 Cart sensor Cartridge Wait Transfer to Flask gas Flush Transfer Line Del S2 Cart sensor Cartridge Wait Transfer to Flask gas Flush Transfer Line Del S3 Cart sensor Cartridge Wait Transfer to Flask gas Transfer Complete Dry Cart top Del S3 Cart top Cartridge Wait Dry Cart top End Time sec 15 5 5 5 5 5 10 10 60 30 720 15 30 10 30 40 45 0 5 15 10 45 5 15 10 45 5 15 10 45 0 60 15 5 120 Global Time N Zz E 22222 ZZZ SZ Zi ZZjoZ ZZ zz z LL z Cart PL PVDF Total run time 35 45 Protein Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 139 Flush Small Loop Cart 10 N 3 75 Load X1 Cart sm loop 70 Y 4 131 Dry Cart top 30 N 5 139 Flush Small Loop Cart 10 N 6 135 Flush Cart Reagent Block 5 N 7 11 Del R2g Cart top 30 N 8 140 Flush Large Loop Cart 10 N 9 6 Load RI Cart Ig loop 20 N 10 131 Dry C
19. 219 217 220 218 171 213 215 171 213 222 156 166 Function Name Begin Wash Small Loop Cart Wash Large Loop Cart Wash Cart Reagent Block Wash Cart Solvent Block Wash Input Block S2 Wash Input Block S3 Wash Output Block S2 Wash Output Block S3 Flush Small Loop Cart Flush Large Loop Cart Flush Cart Reagent Block Flush Input Block Flush Output Block Flush Cart Solvent Block Backflush R1 Backflush R2g Backflush R3 Backflush R3g Backflush S1 Backflush S2 Backflush S3 Backflush X1 Backflush X1g Backflush X3 Cart Wash Small Loop Flask Flush Small Loop Flask Wash Large Loop Flask Flush Large Loop Flask Del S4 Flask Dry Flask Empty Flask Del S4 Flask Dry Flask Flush Flask Injector Backflush R4 Backflush R5 Global Time sec Time 0 20 20 20 20 15 15 15 15 60 60 60 60 60 60 20 20 60 20 20 20 20 60 20 60 15 60 15 60 15 10 20 10 10 60 20 20 Z Z Z z Z Zz Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Procedure Listings C 45 Post Run Valve Block Wash X3 C 46 Procedure Listings Step 38 39 40 41 42 Function 176 186 196 206 259 Total run time 12 56 Step O J O asa 0 PM 0 0 M PD N M M MM MMM amp a a 4 4 a 4 so O O O N O d Ss 0 ND O O O JO G ON O Function 258 303 304 305 215 201 94 116 117 257 136 140 135 137 141 212 213 215 201 217 218 213 226 285
20. 258 303 304 77 79 186 166 305 310 257 308 257 307 317 310 257 318 164 310 Function Name Set Tolerance 100th psi Wait Compare Saved Pressure Vent R4 Set Tolerance 100th psi Compare Pressures 10th psi Set Reg Setpoint 10th psi Wait Close Pressure Valve Vent S4 Compare Pressures 10th psi Restore Reg Setpoint Del R4 Waste Flush Large Loop Flask Wash Large Loop Flask Flush Large Loop Flask End Function Name Begin Select Regulator Save Regulator Setpoint Vent X1 Backflush X1 Backflush X2 Backflush R5 Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Vent R5 Set Tolerance 100th psi Time sec 5 30 0 15 10 0 38 30 1 25 0 0 10 10 20 30 0 Time sec 0 7 0 5 10 10 5 38 20 40 1 15 38 0 5 30 0 15 10 Global Time N Zz ZZ Z zz z ZZZ Z Z Z Z ZZ Global Time Z ZZ Z ZZ ZZ ZZZo ZZ Zz Z Zz Zz zz Global Step Function Function Name Time sec Time 20 307 Compare Pressures 10th psi 0 N 21 305 Set Reg Setpoint 10th psi 38 N 22 257 Wait 30 N 23 308 Close Pressure Valve 1 N 24 77 Vent X1 15 N 25 307 Compare Pressures 10th psi 0 N 26 305 Set Reg Setpoint 10th psi 38 N 27 257 Wait 30 N 28 308 Close Pressure Valve 1 N 29 184 Vent X2 25 N 30 307 Compare Pressures 10th psi N 31 30
21. 307 305 28 24 136 144 136 309 259 S1 S2 S3 Leak Test Total run time 7 45 C 36 Procedure Listings Step AN DU FWD 11 12 13 14 15 16 Function 258 303 304 47 49 59 69 305 310 257 308 257 307 317 310 257 Function Name Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Vent R3 Set Tolerance 100th psi Compare Pressures 10th psi Set Reg Setpoint 10th psi Flush R3 Del R3 Waste Flush Cart Solvent Block Wash Cart Solvent Block Flush Cart Solvent Block Restore Reg Setpoint End Function Name Begin Select Regulator Save Regulator Setpoint Vent S1 Backflush S1 Backflush S2 Backflush S3 Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Time sec 20 30 1 15 38 0 5 30 0 15 10 0 15 5 60 10 10 30 Time sec 0 4 0 5 10 10 10 38 20 40 1 15 38 30 Global Time N Zz Z Z Z ZZZ Z Z Z ZZZ Global Time N ZZ Z ZE zi ZZ E z zz zz Step 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Function 318 47 310 307 305 257 308 57 307 305 257 308 67 307 309 48 44 136 64 136 54 136 259 R4 S4 Leak Test Total run time 4 35 Step AN O
22. 36 37 15 40 46 10 17 18 19 20 34 35 36 37 16 15 46 1 11 15 46 15 16 23 46 7 11 15 16 46 10 15 40 46 7 11 15 22 46 7 11 15 21 46 15 10 38 45 1 11 12 12 16 23 7 11 12 22 7 11 12 21 17 18 19 20 34 35 36 37 10 12 38 45 28 32 45 28 30 28 31 Global Value 0 0 0 o O O O O O O O O O O O O O O O O O O O O O O O O O O O Sensor Function N N Y ZZ Zz Z ZZ ZZ Zz Zz z lt Z ZZ Z Z Z Z Z ZZ E Z lt Global Time Y N Y lt lt lt lt lt Z Z Z Z Z Z lt lt x lt lt lt Z lt lt ZZZ Z zz lt lt lt lt lt K lt Function Listing A 5 Number 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 A 6 Function Listing Reagent R4 R4 R4 R4 R4 R4 R4 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5 54 54 54 54 54 54 54 54 S4 S4 X2 X2 X2 X2 X2 X2 X2 X2 X2 X2 X2g X2g Name Vent R4 Flush R4 Backflush R4 Del R4 Waste Reserved Reserved Reserved Del R5 Flask Load R5 Flask sm loop Load R5 Flask Ig loop Vent R5 Flush R5 Backflush R5 Del R5 Waste Reserved Reserved Reserved Del S4 Flask Load S4 Flask sm loop Load S4 Flask lg loop Vent S4 Flush S4 Backflush S4 Del S4 Waste Reserved Reserved Reserved D
23. 60 20 0 10 20 0 N ZZ ZZ ZZ ZZ LIL Z Z Zz ZZ Zz ZZ ZZ Z Z z z Z Procedure Listings C 5 Bottle Change for R4 Total run time 1 35 Step O MN OA ADN gt A N O Function 258 264 303 154 156 260 155 157 218 220 218 259 Bottle Change for Total run time 3 00 R4 Leak C 6 Procedure Listings Step O N OA FR WD ND N N Aa do ss oa N O O JO an ON Function 258 264 303 304 154 156 260 305 310 257 308 257 307 317 310 257 318 309 155 157 218 220 Function Name Begin Set for Bottle R4 Select Regulator Vent R4 Backflush R4 Pause for Bottle Change Flush R4 Del R4 Waste Flush Large Loop Flask Wash Large Loop Flask Flush Large Loop Flask End Function Name Begin Set for Bottle R4 Select Regulator Save Regulator Setpoint Vent R4 Backflush R4 Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Restore Reg Setpoint Flush R4 Del R4 Waste Flush Large Loop Flask Wash Large Loop Flask Time sec 0 0 6 10 20 0 15 10 10 10 20 0 Time sec 0 0 6 0 10 20 0 38 20 40 0 15 38 0 5 30 0 0 15 10 10 10 Global Time Z Z Z Ziz Z Z Z ZiZz ZZ Global Time Z Z Z Z ZZ Z 2 22 ZZ Zz Z Zz Z Z Z ZIZ Step 2
24. 7 6 PTH Separation the altered gradient will be downloaded to the 140C pump To make changes to an active gradient Step Action 1 Select the Gradient screen and select gradient Fast Normal 1 from the pull down list 2 Highlight the time line that requires the change and alter the value in the Time box and or B box at the top of the column 3 To insert or delete row highlight the time line and click the Insert Row or Delete Row button Alter the time and B as desired 4 To save the changes pull down the File menu from the top menu bar and select Save Gradient IMPORTANT By selecting Save Gradient from the File menu the current fast gradient will be altered and the changes sent to the sequencer if it is active To save the new gradient without modifying the original fast gradient select Save As from the File menu and enter the new gradient name Optimizing the Chromatography Flattening the Baseline Reducing Negative Baseline Slope at the Start of the Chromatogram In order to achieve a high sensitivity sequence it is critical to minimize any baseline rise One factor which causes the baseline rise in PTH chromatograms is the slightly higher absorbance properties of solvent B2 Eliminating this problem can increase accuracy in computer integration at high sensitivity Acetone has a very high UV absorbance at 269 nm a wavelength that is optimal for PTH amino acid analysis When small amo
25. 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 B 10 Cycle Method and Gradient Listings Function 140 138 144 143 146 107 137 136 111 64 135 137 140 136 138 257 131 142 127 141 63 148 121 141 53 148 121 141 63 148 121 128 131 61 148 131 259 Function Name Flush Large Loop Cart Flush Output Block Wash Cart Solvent Block Wash Cart Reagent Block Wash Large Loop Cart Wash Output Block S2 Flush Input Block Flush Cart Solvent Block Wash Input Block S3 Del S3 Waste Flush Cart Reagent Block Flush Input Block Flush Large Loop Cart Flush Cart Solvent Block Flush Output Block Wait Dry Cart top Set Cart Temperature Ready Transfer to Flask Flush Transfer Line Del S3 Cart sensor Cartridge Wait Transfer to Flask gas Flush Transfer Line Del S2 Cart sensor Cartridge Wait Transfer to Flask gas Flush Transfer Line Del S3 Cart sensor Cartridge Wait Transfer to Flask gas Transfer Complete Dry Cart top Del S3 Cart top Cartridge Wait Dry Cart top End Time sec 10 10 10 15 10 15 10 10 5 5 40 30 30 60 30 0 40 45 0 5 15 10 45 5 15 10 45 5 15 10 45 0 60 15 5 120 Global Time N Zz Z gt Zizi ZZ Z Zz ZZ 2 2 2 2 2 Z 2 ZZ lt lt ZZZ gt 2 2 Z Z Z 2 Z 2 ZZZ Cart Gas phase Total run time 41 05 Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 137 Flush Input Bloc
26. Peptide None None Cart Begin Gas phase 35 None 35 None 35 None 35 None 45 Flask Optimize Cart Flask Temp C 64 Flask Normal Flask Prep Pump Flask Blank Flask Standard 64 Run Gradient 64 Flask Standard 64 Injector Optimize 64 Manual Injection 64 Flask Optimize Flsk Cycle Method and Gradient Listings B 33 Column Temp C 55 Fast Normal Prep Pump Fast Normal Fast Normal 55 Fast Normal 55 Fast Normal 55 Fast Normal 55 Fast Normal 55 Fast Normal Gradient List Gradient Tables Fast Normal 1 Gradient Gradient Name Max Pressure Fast Normal 1 Min Pressure Target Pressure Target Time Data Collect Time Prep Pump Gradient Gradient Name Max Pressure Prep Pump Min Pressure Target Pressure Target Time Data Collect Time B 34 Cycle Method and Gradient Listings 4000 1000 1 0 20 0 4000 1500 1 0 10 0 Time min 0 3 0 4 18 18 5 19 21 5 Time min 20 B 16 45 90 90 90 B 50 50 HL min 325 325 325 325 325 325 325 HL min 325 325 Events 12 O Aa gt da da Ja Events 0 0 Procedure Listings Overview About This All available procedure lists are in this appendix Appendix In This Chapter This appendix contains the following topics Topic See Page Bottle Change Procedure List C 2 Cleanup Procedure List C 17 Electrical T
27. X X3 system clean out 5 8 Index 5 Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com www appliedbiosystems com s AS Applied Na Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 05 2002 Part Number 4314375B an Applera business
28. e g safety glasses gloves or protective clothing After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Site Preparation and Safety Guide About MSDSs Ordering MSDSs A site preparation and safety guide is a separate document sent to all customers who have purchased a Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and waste profiles Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer When hazards exist warnings are prominently displayed on the labels of all chemicals Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update MSDSs provide you with the safety information you need to store handle transport and dispose of the chemicals safely We strongly recommend that you replace the appropriate MSDS in your files each time you receive a new MSDS packaged with a hazardous chemical NA Nile CHEMICAL HAZARD Be sure to familiarize yourself with the MSDSs before using reagents or solvents You can order free additional copies of MSDSs for chemicals manufactured
29. end step 3 6 flask load guidelines 6 13 listing of A 1 to A 11 Ready to Receive 3 6 sensor 3 6 synchronization 3 6 table of function numbers 3 6 numbering format 3 6 required functions 3 7 valve controlling 3 6 G Gas phase method 3 11 B 32 global time setting and activating 4 3 glutamic acid 7 10 modifed 6 16 glutamic acid separating 7 9 glutamine separating 7 5 glycine separating 7 5 GP PVDF Peptide method 3 12 B 33 GP PVDF Protein method 3 12 B 32 gradient defininga 3 2 fast 7 5 table of name conditions B 34 H help e mail address D 1 Internet address D 5 regional sales offices D 3 telephone hours D 1 telephone fax U S D 2 histidine 7 8 HPLC programming fast gradient 7 5 I Inject Optimize method 3 13 B 33 injecting optimizing 6 2 to 6 3 injection optimizing 6 20 sample percentage 6 21 Internet address Documents on Demand D 5 ion pairing additive 7 4 isoleucine from lysine 7 10 L lag defining 6 15 leak procedures C 32 to C 44 leak test passing specs 5 6 leak test procedures cartridge A C 32 cartridge B C 32 cartridge block C 42 cartridge C C 33 cartridge D C 33 cartridge reagent block C 41 flask input C 43 flask leak C 43 R1 C 34 R2 C 35 R3 C 35 R4 84 C 37 regulator 10 C 41 regulator 9 C 40 1 S2 S3 C 36 waste system C 44 X3 C 39 leucine separating 7 10 loop cartridge volume loading 6 10 flask volume loading 6 12 lysine separating from leucine 7 10 lysine from iso
30. particularly proline proceeds more slowly than others and will benefit from an extended cleavage time or increased cleavage temperature Cleavage for proline residues can be extended up to 600 seconds twice as long as a standard cleavage Alternatively the temperature of the cleavage can be increased to 55 C These extreme cleavage conditions should be used only where needed as the rate of sample degradation would be significantly increased if these conditions were used for every cycle Gas phase cleavage is performed by delivering TFA vapor through the active cartridge for a prescribed period of time in order for the cleavage to take place Gas phase cleavage requires more time than pulsed liquid phase cleavage and as a result the standard gas phase cycles are approximately five minutes longer than their pulsed liquid counterparts For the best results using the gas phase cycles it may be necessary to reduce the R3 pressure setting to 0 8 1 0 psi Too high a TFA flow rate through the cartridge will result in higher than expected lag If the sequencing lag per cycle for a sample using the gas phase cleavage cycle is higher than when using the pulsed liquid cycle then the R3 regulator pressure should be reduced Gas phase cleavage cycles tend to be somewhat cleaner than pulsed liquid cycles that is the level of chemistry artifact peaks is usually slightly lower Gas phase cleavage may help reduce washout of hydrophobic peptides Drying af
31. 258 Begin 0 N 2 235 Set as Residue Cycle 0 N 3 218 Flush Large Loop Flask 10 N 4 173 Load S4 Flask lg loop 15 N 5 213 Dry Flask 10 N 6 218 Flush Large Loop Flask 10 N 7 228 Ready to Receive 1 N 8 213 Dry Flask 15 N 9 236 Pre Conversion Dry 0 Y 10 218 Flush Large Loop Flask 10 N Cycle Method and Gradient Listings B 27 Step 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Function 153 213 218 173 218 257 237 226 227 213 173 213 218 173 213 218 221 257 221 213 238 257 225 249 171 213 212 215 171 213 212 222 221 259 Run Gradient Total run time 31 32 Step 1 2 B 28 Cycle Method and Gradient Listings Function 258 257 Function Name Load R4 Flask Ig loop Dry Flask Flush Large Loop Flask Load S4 Flask Ig loop Flush Large Loop Flask Wait Post Conversion Dry Load Position Prepare Pump Dry Flask Load S4 Flask Ig loop Dry Flask Flush Large Loop Flask Load S4 Flask lg loop Dry Flask Flush Large Loop Flask Flush Injector Wait Flush Injector Dry Flask Concentrate Sample Wait Load Injector Inject Pos Collect Data Del 54 Flask Dry Flask Bubble Flask Empty Flask Del 54 Flask Dry Flask Bubble Flask Flush Flask Injector Flush Injector End Function Name Begin Wait Time sec 20 10 10 15 10 540 0 450 15 10 10 15 10 10
32. 3 11 Del R2g Cart top 30 N 4 140 Flush Large Loop Cart 10 N 5 6 Load R1 Cart Ig loop 20 N 6 131 Dry Cart top 30 N 7 140 Flush Large Loop Cart 5 N 8 135 Flush Cart Reagent Block 5 N 9 11 Del R2g Cart top 170 N 10 132 Dry Cart bottom 30 N 11 140 Flush Large Loop Cart 5 N 12 6 Load R1 Cart Ig loop 20 N 13 131 Dry Cart top 30 N 14 140 Flush Large Loop Cart 5 N 15 135 Flush Cart Reagent Block 5 N 16 11 Del R2g Cart top 170 N 17 132 Dry Cart bottom 30 N 18 140 Flush Large Loop Cart 5 N 19 6 Load RI Cart Ig loop 20 N 20 131 Dry Cart top 30 N Cycle Method and Gradient Listings B 17 Step 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 B 18 Cycle Method and Gradient Listings Function 140 135 11 146 111 140 137 136 132 142 63 148 61 148 51 148 51 148 131 53 148 51 148 51 148 61 148 131 137 140 26 30 136 140 138 144 143 146 107 137 136 Function Name Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Wash Large Loop Cart Wash Input Block S3 Flush Large Loop Cart Flush Input Block Flush Cart Solvent Block Dry Cart bottom Set Cart Temperature Del S3 Cart sensor Cartridge Wait Del S3 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Dry Cart top Del S2 Cart s
33. 305 303 305 303 305 303 Function Name Load Position Del X3 Flask Dry Flask Flush Flask Injector Backflush R1 Backflush R2g Backflush R3 Backflush S1 Backflush S2 Backflush S3 Backflush X1 Vent X3 Cart Backflush X3 Cart Backflush X3 Flask Backflush R4 Backflush R5 Backflush S4 Backflush X2 Backflush X2g End Function Name Begin Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Time sec 0 30 5 10 10 10 10 10 10 10 10 5 10 10 15 10 10 10 10 0 Time sec 0 1 20 2 10 3 30 4 17 5 50 6 35 7 20 8 Global Time N Zz z Z Z ZZ ZZzZZ Zz ZZ Z Z Z ZZ z Global Time N ZIZ Z ZZ ZZ Z ZZ ZZ ZZ Z Step 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Function 305 226 9 19 29 39 49 59 69 79 89 99 206 156 166 176 186 196 135 136 123 121 132 124 121 125 131 126 131 147 137 138 139 140 214 217 218 222 223 222 215 Function Name Set Reg Setpoint 10th psi Load Position Backflush R1 Backflush R2g Backflush R3 Backflush R3g Backf
34. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 Function 121 124 53 131 125 63 131 126 63 131 147 212 215 221 152 213 217 162 213 217 202 213 217 182 213 217 215 218 173 213 218 173 213 218 221 257 225 222 259 Function Name Transfer to Flask gas Select Cartridge B Del S2 Cart Sensor Dry Cart top Select Cartridge C Del S3 Cart Sensor Dry Cart top Select Cartridge D Del S3 Cart Sensor Dry Cart top End Cartridge Select Bubble Flask Empty Flask Flush Injector Load R4 Flask sm loop Dry Flask Flush Small Loop Flask Load R5 Flask sm loop Dry Flask Flush Small Loop Flask Load X3 Flask sm loop Dry Flask Flush Small Loop Flask Load X2 Flask sm loop Dry Flask Flush Small Loop Flask Empty Flask Flush Large Loop Flask Load S4 Flask Ig loop Dry Flask Flush Large Loop Flask Load S4 Flask Ig loop Dry Flask Flush Large Loop Flask Flush Injector Wait Load Injector Flush Flask Injector End Time sec 45 1 13 60 1 13 60 1 13 60 20 20 18 10 18 10 35 10 35 10 10 10 18 10 18 10 20 10 20 Global Time N Z Z er Z Z ZZ Zz Z ZZ ZZ 2 22 223232 ZZ ZZ Z eZ Z Z Z Z Z LL Z Idle Procedure Idle Total run time 1 33 Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 8 Flush R1 5 N
35. 50 methanol from the X1 bottle to the cartridge during coupling to improve the repetitive yield GP PVDF Protein sequence a protein sample that has been applied to PVDF membrane by electroblotting or using a ProSorb device The GP PVDF Protein method includes A begin cycle to prepare the sample for sequencing A blank cycle A PTH amino acid standard cycle Sequencing cycles The GP PVDF Protein method delivers TFA vapor to the cartridge for cleavage of the ATZ amino acid and includes a delivery of 50 methanol from the X1 bottle to the cartridge during coupling to improve the repetitive yield PL PVDF Peptide sequence a peptide sample that has been applied to PVDF membrane using a ProSorb device The PL PVDF Peptide method includes A begin cycle to prepare the sample for sequencing 9 A blank cycle A PTH amino acid standard cycle Sequencing cycles The PL PVDF Peptide method delivers a small volume of liquid TFA to the cartridge for cleavage of the ATZ amino acid A few microliters of BiobrenePlus solution diluted 1 10 with methanol should be applied to the sample membrane before sequencing GP PVDF Peptide sequence a peptide sample that has been applied to PVDF membrane using a ProSorb device The GP PVDF Peptide method includes A begin cycle to prepare the sample for sequencing 9 A blank cycle A PTH amino acid standard cycle Sequencing cycles The GP PVDF Peptide method delive
36. 799 7777 41 0 41 790 0676 The Netherlands Nieuwerkerk a d IJssel 31 0 180 331400 31 0 180 331409 United Kingdom Warrington Cheshire 44 0 1925 825650 44 0 1925 282502 All other countries not listed 44 0 1925 282481 44 0 1925 282509 Warrington UK Japan Japan Hacchobori Chuo Ku Tokyo 81 3 5566 6230 81 3 5566 6507 Getting Help D 3 D 4 Getting Help Region Telephone Dial Fax Dial Lat in America Del A Obregon Mexico 305 670 4350 305 670 4349 To Reach Technical We strongly encourage you to visit our Web site for answers to frequently asked Support Through Questions and for more information about our products You can also order technical the Internet documents or an index of available documents and have them faxed or e mailed to you through our site The Applied Biosystems Web site address is http www appliedbiosystems com techsupp To submit technical questions from North America or Europe Step Action 1 Access the Applied Biosystems Technical Support Web site 2 Under the Troubleshooting heading click Support Request Forms then select the relevant support region for the product area of interest 3 Enter the requested information and your question in the displayed form then click Ask Us RIGHT NOW blue button with yellow text 4 Enter the required information in the next form if you have not a
37. 97 99 206 260 305 310 257 308 257 307 317 310 257 318 309 98 94 Function Name Select Regulator Vent X3 Cart Backflush X3 Cart Backflush X3 Flask Pause for Bottle Change Flush X3 Cart Del X3 Waste Flush Cart Reagent Block Wash Cart Reagent Block Flush Cart Reagent Block Del X3 Waste Flask Flush Large Loop Flask Wash Large Loop Flask Flush Large Loop Flask End Function Name Begin Set for Bottle X3 Select Regulator Save Regulator Setpoint Vent X3 Cart Backflush X3 Cart Backflush X3 Flask Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Restore Reg Setpoint Flush X3 Cart Del X3 Waste Global Time sec Time 8 10 60 60 0 15 60 10 10 20 60 10 10 20 0 N Z Z Z Z Z Z Z Z Z Z Z Z Z Z Global Time sec Time 0 0 8 0 10 60 60 0 38 20 40 0 15 38 30 15 60 N Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z 2 Procedure Listings C 15 C 16 Procedure Listings Step 22 23 24 25 26 27 28 29 Function 135 143 135 207 218 220 218 259 Function Name Flush Cart Reagent Block Wash Cart Reagent Block Flush Cart Reagent Block Del X3 Waste Flask Flush Large Loop Flask Wash Large Loop Flask Flush Large Loop Flask End Time sec 10 10 20 60 10
38. B2 solvent 7 3 background amino acid 6 15 baseline adding phosphate ion 7 7 minimizing 7 7 begin step function 258 3 6 BioBrene Plus P N 1 6 treating with 2 2 Blott cartridge 2 2 bottle change 5 9 to 5 10 process describing the 5 9 bottle change procedures 5 10 C 2 to C 16 R1 C 2 R1 leak C 2 R2 C 3 R2 leak C 4 R3 C 4 R3 leak C 5 R4 C 6 R4 leak C 6 R5 C 7 R5 leak C 7 S1 C 8 S1 leak C 8 S2 C 9 S2 leak C 9 S3 C 10 S3 leak C 10 S4 C 11 S4 leak C 11 X1 C 12 X1 leak C 13 X2 C 13 X2 leak C 14 X3 both C 14 X3 both leak C 15 bottle leak test 5 6 bubbles detecting 6 9 butyl chloride see also S3 2 4 C cartridge cycle procedures B 2 to B 24 begin B 2 begin gas phase B 4 precycle B 6 cartridge leak test 5 6 chromatography optimizing 7 7 7 11 the injector 6 2 to 6 3 cleanup defining 3 procedures 5 8 cleanup procedures C 17 to C 24 delivery line backflush C 17 system clean out X3 C 20 system flush argon C 22 cleavage defining 6 18 drying after 6 18 Edman degradation 2 4 efficiency of 6 15 gas phase 6 18 optimizing 6 18 pulsed liquid 6 18 Rx diagram 2 4 conversion optimizing 6 19 coupling base delivery 6 16 defining 6 16 drying after 6 17 Edman degradation 2 3 post coupling wash 6 17 temperature 6 17 customer support e mail address D 1 help D 1 to D 5 Internet address D 5 regional sales offices D 3 telephone fax U S D 2 cycle 3 9 to 3 10 cartridge listing of B 2 to B 24 Index 1 crea
39. Cart top Cartridge Wait Dry Cart top Del S2 Cart sensor Cartridge Wait Del S2 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Del S3 Cart top Cartridge Wait Dry Cart top Flush Input Block Flush Large Loop Cart Load R3 Cart Ig loop Transfer R3 Cart gas Flush Cart Solvent Block Flush Large Loop Cart Flush Output Block Wash Cart Solvent Block Wash Cart Reagent Block Wash Large Loop Cart Wash Output Block S2 Flush Input Block Flush Cart Solvent Block Wash Input Block S3 Del S3 Waste Flush Cart Reagent Block Flush Input Block Flush Large Loop Cart Flush Cart Solvent Block Flush Output Block Wait Dry Cart top Set Cart Temperature Ready Transfer to Flask Flush Transfer Line Del S3 Cart sensor Cartridge Wait Transfer to Flask gas Time sec Global Time N zZz 2 2z Z Z 2 xXx ZZ Z Z Zz Zz ZZ ZZzZzizzzzizmzmzmzzzzzzZzzzzzzz Z Global Step Function Function Name Time sec Time 81 141 Flush Transfer Line 5 N 82 53 Del S2 Cart sensor 15 N 83 148 Cartridge Wait 10 N 84 121 Transfer to Flask gas 45 N 85 141 Flush Transfer Line 5 N 86 63 Del S3 Cart sensor 15 N 87 148 Cartridge Wait 10 N 88 121 Transfer to Flask gas 45 N 89 128 Transfer Complete 0 N 90 131 Dry Cart top 60 N 91 61 Del S3 Cart top 15 N 92 148 Cartridge Wait 5 N 93 131 Dry Cart top 120 N 94 259 End 0 N Cart GP PVDF Total run time 43 05 Protein Global Step Function
40. Cartridge Wait Del S2 Cart top Cartridge Wait Dry Cart top Del S2 Cart sensor Cartridge Wait Del S2 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Del S3 Cart top Cartridge Wait Dry Cart top Time sec 5 170 30 5 20 30 5 5 170 30 5 20 30 5 5 170 10 10 15 20 30 60 48 15 10 10 10 5 Global Time N Z ZZ Z Ziz Z Z Z Zz Z ZZZzZ ZZ Zz Z ZZ Z ZZ ZzZZZZZizZzZzizZ Z Zz Z Z z Z Global Step Function Function Name Time sec Time 49 34 Del R3g Waste 30 N 50 31 Del R3g Cart top 720 N 51 143 Wash Cart Reagent Block 15 N 52 135 Flush Cart Reagent Block 30 N 53 144 Wash Cart Solvent Block 10 N 54 136 Flush Cart Solvent Block 30 N 55 131 Dry Cart top 40 N 56 142 Set Cart Temperature 48 N 57 127 Ready Transfer to Flask 0 N 58 141 Flush Transfer Line 5 N 59 63 Del S3 Cart sensor 15 N 60 148 Cartridge Wait 10 N 61 121 Transfer to Flask gas 45 N 62 141 Flush Transfer Line 5 N 63 53 Del S2 Cart sensor 15 N 64 148 Cartridge Wait 10 N 65 121 Transfer to Flask gas 45 N 66 141 Flush Transfer Line 5 N 67 63 Del S3 Cart sensor 15 N 68 148 Cartridge Wait 10 N 69 121 Transfer to Flask gas 45 N 70 128 Transfer Complete 0 N 71 131 Dry Cart top 60 N 72 61 Del S3 Cart top 15 N 73 148 Cartridge Wait 5 N 74 131 Dry Cart top 120 N 75 259 End 0 N Flask Optimize Cart Total run time 5 25 Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 1
41. Premix will usually give a good separation with His and Arg in these later elution positions Histidine If Then His coelutes with Ala increase the buffer concentration His needs to move before Ala add an additional 5 mL of Premix buffer concentrate per liter of Solvent A3 Arginine Then Arg coelutes with Tyr increase the buffer concentration Arg needs to move before Tyr a add an additional 5 mL of Premix buffer concentrate per liter of solvent A3 b Increase the B at 0 4 minutes to 14 16 7 8 PTH Separation Pyridylethyl Cysteine The Acidic Amino Acids Aspartic Acid If Then the Ser coelutes with the Arg peak improve the separation by shifting the Ser peak before Arg by a Decreasing the column temperature 2 5 C Adjust the temperature carefully because the separation of Met from Val is reduced by decreasing the column temperature b Decreasing the flow rate A decrease in flow rate from 325 mL min 300 mL min moves Arg slightly relative to Ser without significantly impacting any other separations The B at 18 minutes should be decreased by 1 to maintain optimum separation of lle Lys If Then PECys coelutes with Pro increase the buffer concentration Add approximately 5 mL of Premix buffer concentrate per liter of Solvent A3 to move PECys before Pro The acidic ami
42. S3 5 N 63 64 Del S3 Waste 5 N 64 135 Flush Cart Reagent Block 40 N 65 137 Flush Input Block 30 N 66 140 Flush Large Loop Cart 30 N 67 136 Flush Cart Solvent Block 60 N 68 138 Flush Output Block 30 N Cycle Method and Gradient Listings B 23 Step 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 B 24 Cycle Method and Gradient Listings Function 257 131 142 127 141 63 148 121 141 53 148 121 141 63 148 121 128 131 61 148 131 259 Function Name Wait Dry Cart top Set Cart Temperature Ready Transfer to Flask Flush Transfer Line Del S3 Cart sensor Cartridge Wait Transfer to Flask gas Flush Transfer Line Del S2 Cart sensor Cartridge Wait Transfer to Flask gas Flush Transfer Line Del S3 Cart sensor Cartridge Wait Transfer to Flask gas Transfer Complete Dry Cart top Del S3 Cart top Cartridge Wait Dry Cart top End Time sec 0 40 45 0 5 15 10 45 5 15 10 45 5 15 10 45 0 60 15 5 120 Global Time Y DE LL i ge AZ ZZZ Flask Cycle List Flask Blank Total run time 35 57 Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 233 Set as Blank Cycle 0 N 3 171 Del 54 Flask 15 N 4 213 Dry Flask 10 N 5 215 Empty Flask 20 N 6 151 Del R4 Flask 15 N 7 213 Dry Flask 10 N 8 215 Empty Flask 20 N 9 218 Flush Large Loop Flask 10 N 10 173 Load SA Flask Ig loop 15 N 11 213 Dry Flask 10 N 12 218 F
43. S4 15 N 8 177 Del S4 Waste 10 N 9 218 Flush Large Loop Flask 20 N 10 259 End 0 N Bottle Change for Total run time 2 40 S4 Leak Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 269 Set for Bottle S4 0 N 3 303 Select Regulator 6 N 4 304 Save Regulator Setpoint 0 N 5 174 Vent S4 10 N 6 176 Backflush S4 20 N 7 260 Pause for Bottle Change 0 N Procedure Listings C 11 Bottle Change for X1 C 12 Procedure Listings Step 10 11 12 13 14 15 16 17 18 19 20 21 22 Function 305 310 257 308 257 307 317 310 257 318 309 175 177 218 259 Total run time 3 25 Step AN Asa WD gt a 0 Ww N O Function 258 270 303 77 79 89 260 78 74 84 143 135 259 Function Name Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Restore Reg Setpoint Flush S4 Del S4 Waste Flush Large Loop Flask End Function Name Begin Set for Bottle X1 Select Regulator Vent X1 Backflush X1 Backflush X1g Pause for Bottle Change Flush X1 Del X1 Waste Del X1g Waste Wash Cart Reagent Block Flush Cart Reagent Block End Time sec 38 20 40 0 15 38 30 15 10 20 Time sec 0 0 7 10 60 20 0 15 60 10 10 20 0 Global Time N Z ZZ Z Z Z ZIZ Z Z ZZZ Global Time N Zz Zz zz z ZZ
44. Saved Pressure Open Valves 24 32 45 Set Tolerance 100th psi Global Time sec Time 0 8 0 38 20 5 1 10 38 0 30 10 0 10 10 0 0 10 0 Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Global Time sec Time 0 8 0 38 20 60 1 10 38 0 30 5 0 10 10 Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Procedure Listings C 43 Step 16 17 18 19 Function 307 309 296 259 Waste System Test Total run time 4 30 C 44 Procedure Listings Step AN DU FWD 11 12 13 14 Function 258 303 304 299 310 308 77 307 317 77 310 318 309 259 Function Name Compare Pressures 10th psi Restore Reg Setpoint Open Valves 24 32 45 End Function Name Begin Select Regulator Save Regulator Setpoint Open Valves 49 57 59 Set Tolerance 100th psi Close Pressure Valve Vent X1 Compare Pressures 10th psi Save Regulator Pressure Vent X1 Set Tolerance 100th psi Compare Saved Pressure Restore Reg Setpoint End Time sec 0 0 10 0 Time sec 0 7 0 120 15 1 30 25 0 120 10 Global Time N N N N Global Time N Z Z Z Z Z Z ZZ Z Zz Z Z Shutdown Procedure List Short Term Total run time 21 35 Shutdown Step AN Oa R 0 PM WU 0 0 OOO 0 NP PN N PD PD M MMM N a a 44 4 a 4 0 NO TOF N O O UN DOA A 0 ON O 0 O JO O BR O N O Function 258 145 146 143 144 106 111 107 112 139 140 135 137 138 136 9 19 29 39 49 59 69 79 89 99
45. The timing for transfer from cartridge to flask is defined by a set of steps in the cartridge cycle and one step in the flask cycle A Ready to Receive Function 228 step in the flask cycle indicates that the flask is ready to accept sample transfer from 3 6 Valves Functions Cycles and Methods Other System Functions the cartridge The flask will wait at this step with the cycle timer incrementing until the transfer is complete The beginning and end of the transfer from the cartridge to the flask are defined in the cartridge cycle respectively by the steps Ready Transfer to Flask Function 127 and Transfer Complete Function 128 Synchronization is set up in such a way that the Ready to Receive step in a flask cycle occurs 5 seconds before the Ready to Transfer step in the cartridge cycle Table 3 3 Necessary Cartridge Functions Function Name Number Description Set Cart 142 Used to adjust the cartridge temperature at a Temperature fixed time during a cycle Acceptable temperature range ambient to 70 C Table 3 4 Necessary Flask Functions Function Name Number Description Load Position 226 Switches the sample loop out of the HPLC flow path During a flask cycle this function must precede the Load Injector step in order for the sample loop to be flushed and the sample in the flask to be transferred into the sample loop Set Flask Temperature 230 Used to adjust the flas
46. Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z B 7 Step 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 Function 148 131 140 26 30 136 144 146 140 136 131 53 51 148 51 148 51 148 131 63 131 63 131 259 Cart Pulsed liquid Total run time 33 45 Step AN DU FWD 11 12 B 8 Cycle Method and Gradient Listings Function 258 137 11 140 6 131 140 135 11 132 140 6 Function Name Cartridge Wait Dry Cart top Flush Large Loop Cart Load R3 Cart Ig loop Transfer R3 Cart gas Flush Cart Solvent Block Wash Cart Solvent Block Wash Large Loop Cart Flush Large Loop Cart Flush Cart Solvent Block Dry Cart top Del S2 Cart Sensor Del S2 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Dry Cart top Del S3 Cart sensor Dry Cart top Del S3 Cart Dry Cart top End sensor Function Name Begin Flush Input Block Del R2g Cart top Flush Large Loop Cart Load R1 Cart Ig loop Dry Cart top Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Dry Cart bottom Flush Large Loop Cart Load R1 Cart Ig loop Time sec 5 30 10 50 5 15 10 10 30 30 30 20 15 5 15 5 15 5 30 20 30 20 120 Time sec 0 5 30 10 20 30 5 5 170 30 5 20 Global Time N ZZ ZZZ ZZZ ZZ Z Global Time Z Z Z ZE Z ZZ ZZ Z Z
47. a reagent is changed load times must be changed Cartridge Load 25 Well Function Time Note If the protein sequencer has not been run since the last cold start use the Test view to select and run the Init Sensor procedure Then allow the procedure to run to completion To determine the amount of time necessary for a cartridge load function Step Action 1 From the Pressures and Temperatures view set the desired delivery pressure for the bottle position to be used If the reagent or solvent is not loaded on the instrument perform the bottle change procedure for that bottle position From the Manual Control view select Function 139 Flush Large Loop or Function 140 Flush Small Loop from the cartridge function list Activate the appropriate flush function for 20 seconds to make sure that the loop is clear of any liquid a Activate the load function for the bottle and loop of choice For example select Function 183 to load the large loop with reagent or solvent from the X2 bottle position b Watch for the appearance of a check mark next to the reaction flow sensor field at the top of the screen c Note the elapsed time and add 5 10 seconds Enter this load time in the cycle for this function d Select All Off before leaving the Manual Control view From the Function view enter the load time in the global time field for that function Once the time for loading the large loop ha
48. argon gas to each reagent solvent bottle at a user selectable level See Chemical Warnings Table on page 1 7 for warnings about argon gas To execute the idle procedure Step Action 1 Select Preferences from the Sequencer pull down menu on the upper menu bar 2 Click on the Execute Idle Procedure check box and enter the frequency at which the procedure should be run ranging from once every hour to once every 999 hours 3 Note Itis not possible to pause or cancel an Idle Procedure once it is running The sequencer cannot be programmed until the Idle Procedure run is complete Select the Idle Procedure to be used from the menu and click on the OK button 4 If the sequencer is active when the Idle procedure is selected go to the Start Run view and click on the Update button Init Sensor Procedures About the Init The Procise system uses 11 optical sensors to detect fluid deliveries Every time the Sensor Procedure Start Run button is clicked the Procise system automatically runs the Init sensor procedure This procedure flushes the flow path through each sensor and then takes a dry reading for each sensor If the sensor light path is not completely dry the sensor will not function correctly during sequencing IMPORTANT Always allow the Init Sensor procedure to run to completion If the Procise sequencer has been shut down or if a sensor has been moved or replaced the Init Sensor procedure must
49. be run before sequencing or using manual control functions Whenever a sensor fails to sample sufficient consecutive wet readings before the end of a function the information will be reported in the Event Log Tests and Procedures 5 5 Leak Procedures About Leak Tests Bottle Leak Tests Cartridge Leak Test Flask Leak Test Valve Block Leak Test 5 6 Tests and Procedures There are 18 leak tests or procedures in the Procise system software Bottle and cartridge leak tests can be used routinely IMPORTANT Leak tests alter the pressure settings for reagent solvent and or gas deliveries If a test procedure is interrupted pressure s may remain altered Select the Pressures and Temperature view and click on the Default button to restore the original operating pressures Bottle leak tests can be run directly from the Test view Bottle leak testing is also available from the Bottle Change view by selecting the Bottle Change procedure followed by leak Each bottle leak test reports the following three pressures Pressurization Check that the bottle can be adequately pressurized Monitor Leak Rate Measure the pressure drop with the regulator set to zero Vent Check the venting capability The results of each section of the leak test will be reported in Event Log at the end of the test The actual bottle pressure must be within 0 05 psi of the target pressure in order to pass the leak test The cartridge leak test
50. cycle needs a Begin step and an End step 4 6 Creating Functions Cycles and Methods Creating Methods Copying a Standard To copy the standard method so that it can be edited Methods Step Action 1 Use the View menu to select Sequence Methods View Use the method screen menu to select the method to be copied Use the File menu from the upper menu bar and select Save Method As 2 3 4 Type the new method name and click the OK button Editing the Default To edit the default method Method Step Action 1 Highlight the default method row Select the new cartridge cycle flask cycle and or gradient from each pop up menu To delete a row highlight the row to be deleted and click the Delete button To add a row highlight the row after which the new row will be inserted and click the Insert Row button a Move the cursor to the Cycle ft field and enter the cycle number to be added as an exception b Select the new cartridge cycle flask cycle and or gradient from each pop up menu If the cartridge flask or column temperatures need to be changed move the cursor to the appropriate temperature field and enter the desired temperature Saving an Edited To save the edited method Method Step Action 1 Pull down the File menu from the upper menu bar 2 Select Save Method IMPORTANT Nine exception cycles ar
51. delivery line up to the valve block 8 If necessary load the additional chemicals from the list in the same manner 9 Pull down the File menu from the upper menu bar and select Save Chemicals when the bottle changes are completed 5 10 Tests and Procedures Creating Tests and Procedures Copying a Test or Copy the standard tests and procedures so that they can be edited Procedure To make a copy Step Action 1 Use the View pop up menu to select Cycles and Procedures View 2 Use the upper screen menu to select the type of procedure to be edited 3 Use the lower screen menu to select the specific procedure to be copied 4 Pull down the File menu from the upper menu bar and select Save Cycle Procedures As 5 Type the new procedure name and click on the OK button Editing the Copy To edit the copied procedure Step Action 1 To delete a row highlight the row to be deleted and click on the Delete Row button 2 To insert a row select the function to be inserted from the function list on the left side of the window The function can be selected by either using the scroll down button or typing the function number at the top corner of the function list 3 Highlight the row immediately before the intended insertion point 4 Check off the global box if the global time not is used Type the function time in seconds in the value box Save the Edited To save
52. ee ele eee ei 4 1 Creating User Defined Functions 4 2 Global Times Wu a ee are rs ote av ATH Rana 4 3 Creatina Cycles cuticola Mh ES 4 4 Creating Methods iii ieee skisse So veces Oa 4 7 Tests and Procedures OVEIVIEW geo er eee RR dan gees ea a tn de ane 5 1 Running Testi rareste ee de Pee naw a 5 2 Flow Test Procedures rinite tn dot oe Ee Be hea dode eed 5 3 Startup Procedures o oeio kobe SE ea ee ea eel aes been ay pat 5 4 Idle Proc dures 5 russes ace ce eee eee eed eee ee a dd 5 5 Init Sensor Procedures 5 5 Leak Proc dures 225 idem acne Witla eat i ahaa aa A a 5 6 Shutdown Procedures eii un ee nn alee VENE eee KE 5 7 Cl anup Proc dures smart hi E eee ES AA AA ei 5 8 Electrnical Test Procedure pra ese Ale ON ETS i 5 8 Bottle Change Procedures its ii LA Se ae 5 9 Creating Tests and Procedures 5 11 Chemical Optimization OVErVIEW vaker AGES GREER ERE KRN SEG 6 1 Optimizing the Injector 6 2 Optimizing Flask Dry Times 6 4 Optimizing Sensor Functions 6 7 Sensors and the Event Log 6 8 Optimizing Cartridge Load Functions 6 10 O
53. fluid sensor used in the protein sequencer consists of an infrared emitting diode and photo sensor receiver Fluid is detected by increased light transmission through the Teflon tube due to the change in refractive index Sensor Types The Procise Protein Sequencer uses 11 sensors Cartridge Load 1 Small Sensor Cartridge Load 2 Large Sensor Cartridge A Outlet Sensor Cartridge B Outlet Sensor Cartridge C Outlet Sensor Cartridge D Outlet Sensor Flask Load 1 Small Sensor Flask Load 2 Large Sensor Transfer to Flask Sensor Sample Loop Load Sensor gt gt gt Sample Loop Full Sensor Chemical Optimization 6 7 Sensors and the Event Log Error Message Example of Event Log Entry Interpreting the Event Log Information 6 8 Chemical Optimization If a sensor does not detect fluid during the time allotted for the function an error message will be posted in the Event Log For 9 of the 11 sensors the Procise software will display a dialog box describing the failure and the protein sequencer will pause at the end of the active cycle unless the operator is present to intervene The Transfer to Flask Sensor and the Sample Loop Full Sensor are the exceptions Failure of these sensors to detect fluid results only in the posting of an error in the Event Log An example of the information reported to the Event Log 01 01 1995 4 30 00 PM During step 2 of cycle 1 fluid was not detected by the Cartridge Load 2
54. is finished take off all reagent solvent bottles and empty them Then put empty bottles on the Procise system before running the System Clean out X3 procedure This procedure cleans the entire Procise system Place a bottle of 100 methanol bottle at the X3 bottle position All valve blocks delivery lines reaction cartridges loops injectors and reagent bottles are washed with methanol See Chemical Warnings Table on page 1 7 for warnings about methanol IMPORTANT The System Clean out X3 procedure alters the pressure management system pressure settings When this procedure is finished select the Pressure amp Temperature view and click the Default button to restore the operating pressures This procedure will completely flush and dry all Procise seguencer flow paths with argon See Chemical Warnings Table on page 1 7 for warnings about argon Regulators are reset to default pressure settings during this procedure Electrical Test Procedure 5 8 Tests and Procedures This procedure tests the electrical continuity of key components in the Procise system At step 7 the Rheodyne switches position from load to injection Bottle Change Procedures The Process Two Procedures Starting the Bottle Change Procedure Bottle Change Process Stage Description 1 Starts with back flushing a specific chemical into the reagent bottle The reagent bottle is vented The new bottle is loaded The bottle
55. methanol This procedure does not deliver solvent through the reaction cartridges or to other bottles This procedure is used when the Procise system will be idle for one to two weeks During the short term shutdown procedure ethyl acetate S2B is used to wash all valve blocks delivery lines and loops The flask and injector are washed with S4 After common flow paths are washed and flushed with argon each bottle is briefly backflushed with argon to move reagents out of the Teflon delivery line See Chemical Warnings Table on page 1 7 for warnings about S2B S4 and argon To run a Shutdown procedure from Test view Step Action 1 Select the Test view from the View pop up menu Click on the Shutdown button and select the Shutdown procedure 2 3 Click on the Shutdown button to run the procedure 4 Allow the procedure to run to completion Tests and Procedures 5 7 Cleanup Procedures Three Cleanup Procedures Delivery Line Back flush System Clean out X3 System Flush Argon Three cleanup procedures are programmed in the Procise control software Delivery Line Back flush System Clean out X3 System Flush Argon When the Procise system is to be cleaned first use the delivery line back flush procedure to prepare the system for removal of all reagent bottles This procedure back flushes all reagents and solvents to clean up all delivery lines When this procedure
56. or distributed by Applied Biosystems using the contact information below To order MSDSs Then Over the Internet Go to our web site at www appliedbiosystems com techsupport a Click on MSDSs b Enter keywords or partial words or a part number or the MSDSs Documents on Demand index number c Click on Search d Click on the Adobe Acrobat symbol to view print or download the document or check the box of the desired document and delivery method fax or e mail By automated telephone Use To Obtain Documents on Demand on page D 5 service from any country By telephone in the United Dial 1 800 327 3002 then press 1 States By telephone from Canada To order in Then dial 1 800 668 6913 and English Press 1 then 2 then 1 again French Press 2 then 2 then 1 By telephone from any other See Technical Support on page D 1 country For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer Introduction and Safety 1 3 Instrument Safety Labels About Waste Profiles Before Operating the Instrument Safe and Efficient Computer Use 1 4 Introduction and Safety Safety labels are located on the instrument Each Safety label consists of a Signal Word panel which implies a particular level of observation or action e g CAUTION or WARNING If a safety label encompasses multiple hazards the Signal
57. premix buffer employs an ion pairing additive to improve both peak shape and retention time reproducibility for the PTH derivatives of histidine arginine and the pyridylethyl derivative of cysteine PTH derivatives with positively charged side chain groups interact with underivatized silanol groups on the silica particles in a column causing peak broadening and retention time shifting By adding an ion pairing modifier to the mobile phase the interaction of the basic derivatives with free silanol is significantly reduced through preferential interaction with a strongly acidic ion pairing additive Influence the separation by adding Premix buffer If Then the desired elution order is PTH His before PTH Ala PTH Arg before PTH Tyr and PTH PE Cys before PTH Pro add 25 mL of Premix buffer concentrate P N 401446 to 1 liter of solvent A3 3 5 THF P N 401464 Cap and mix well the PE Cys is not a derivative of interest it is also possible to position His after Ala and Arg after Tyr by using less Premix buffer Approximately 10 mL of Premix will usually give good separation with His and Arg in these later elution positions see Figure 7 2 11 E E 10 o E 9 2 8 E 94 7 6 5 10 15 20 25 Tyrosine Ser Arginine Alanine Histidine DMPTU Glutamic acid 30 35 Premix Concentrate Volume mL Figure 7 2 Effect of Premix Buffer concentration on retention time Grad
58. sec Time 1 258 Begin 0 N 2 303 Select Regulator 5 N 3 304 Save Regulator Setpoint 0 N Procedure Listings C 33 R1 Leak Test C 34 Procedure Listings Step O N Oo A 11 12 13 14 15 16 17 18 19 20 Function 126 131 257 305 310 129 308 129 307 317 310 129 318 309 131 147 259 Total run time 2 55 Step AN DOA R 0 PM a 4 dA dA A on on oa un co O WAN O dd Aa OON O Function 258 303 304 7 9 305 310 257 308 257 307 317 310 257 318 310 307 309 Function Name Select Cartridge D Dry Cart top Wait Set Reg Setpoint 10th psi Set Tolerance 100th psi Pressurize Cart top Close Pressure Valve Pressurize Cart top Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Pressurize Cart top Compare Saved Pressure Restore Reg Setpoint Dry Cart top End Cartridge Select End Function Name Begin Select Regulator Save Regulator Setpoint Vent R1 Backflush R1 Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Vent R1 Set Tolerance 100th psi Compare Pressures 10th psi Restore Reg Setpoint Time sec 0 10 1 35 20 30 1 15 35 0 10 30 Time sec 0 1 0 10 20 38 20 25 1 15 38 30 10 10 Global Time N Zz ZZ ZZ Lr LL ZZZ Global Time Z
59. sequencer cycles While this minimizes unwanted artifact levels excessive S2B delivery may cause extractive loss of some small peptides Chapter 4 Creating Functions Cycles and Methods describes how to create cycles and methods ATZ AA Bae N RI HoN O Va R2 NE CF3COOH o ki R R3 TFA pe R3 O PTC Protein Heal PTC Protein R3 TFA is a highly volatile strong organic acid R3 cleaves the PITC coupled amino acid residue from the amino terminus of the protein thereby producing the anilinothiazolinone ATZ derivative of the amino acid The remaining protein is left with a new amino terminus which will be coupled with PITC in the next cycle S3 butyl chloride and or S2B ethyl acetate S3 butyl chloride and or S2B ethyl acetate are used to extract the cleaved ATZ amino acid from the protein and transfer it from the filter cartridge to the conversion flask Both S3 and S2B are used for extraction in the glass fiber filter cycles and S2B is used for extraction in the Blot PVDF membrane One delivery each of S3 and S2B or three S2B deliveries transfer the ATZ amino acid to the conversion flask The last portion of this solution is transferred to the flask with argon 2 4 Sequence Analysis Chemistry PTH Conversion Diagram Converting with R4A R5 Solubilization and Injection of PTH AAs with S4B A small amount of S4B 20 acetonitrile water is delivered to the flask just before the extraction solution rea
60. side chain carboxylic acid group with aniline The derivative of Asp can be found just before the DPTU peak in the chromatogram the derivative of Glu is just after DPTU The extent of modification of Asp and Glu residues increases slightly with each sequencing cycle The effect is Coupling Temperature Drying After Coupling Post coupling Wash more pronounced for Glu residues The rate of modification of Asp and Glu residues also increases with the coupling temperature and is most noticeable for relatively large gt 100 picomoles amounts of sample The temperature of the cartridge during coupling is set high enough to promote fast efficient reaction of PITC with the amino terminal amino group without excessive side reactions For example the standard pulsed liquid cycles use a coupling temperature of 45 C while the pulsed liquid blot cycles use a 48 C coupling temperature Under the basic conditions necessary for coupling Asp and Glu acid residues are slowly modified by reaction of the side chain carboxylic acid group with aniline The rate of modification of Asp and Glu residues is slightly higher on glass fiber than on PVDF The lower coupling temperature for glass fiber provides a rate comparable to PVDF at the higher temperature The rate of modification of Asp and Glu residues also increases with the length of coupling and is most noticeable for relatively large gt 100 picomoles amounts of sample Drying after coupling p
61. subtracting the Remaining value from the Time value Pre Conversion Dry time Time value Remaining value 105 Calculate the optimum time for the Post Conversion Dry step as follows Post Conversion Dry time Time value Remaining value 250 The result must be a positive number If it is not ensure that the flask is set to the correct temperature and that two full loads of S4 are being delivered to the flask during the procedure Scroll to the Functions dialog box Change the global value of Function 236 Pre Conversion Dry to the optimized value you calculated Change the global value of Function 237 Post Conversion Dry to the optimized value you calculated Chemical Optimization 6 5 Verifying The To set up the protein sequencer for a run Optimized Settings 6 6 Chemical Optimization Step Action 1 Install a reaction cartridge in the cartridge A position on the protein sequencer 2 Perform a leak test on cartridge A 3 Scroll to the Start Run dialog box and configure cartridge A as follows Parameter Setting Cartridge A 1st Filename Your choice Number of cycles 1 Method Flask Optimization To verify the optimization Step Action 1 Click Start Run The Init Sensor procedure begins You can click Jump to advance to the last step of this procedure 2 Once the cartridge and flask have reache
62. support 8 00 a m to 6 00 p m Eastern Time All Other Products 5 30 a m to 5 00 p m Pacific Time Getting Help D 1 To Contact Technical D 2 Support by Telephone or Fax Getting Help In North America To contact Applied Biosystems Technical Support use the telephone or fax numbers given below To open a service call for other support needs or in case of an emergency dial 1 800 831 6844 and press 1 Product or Product Area Telephone Dial Fax Dial ABI PRISM 3700 DNA Analyzer 1 800 831 6844 then press 8 1 650 638 5981 DNA Synthesis 1 800 831 6844 then press 21 1 650 638 5981 Fluorescent DNA Sequencing 1 800 831 6844 then press 22 1 650 638 5981 Fluorescent Fragment Analysis includes GeneScan applications 1 800 831 6844 then press 23 1 650 638 5981 Integrated Thermal Cyclers ABI PRISMO 877 and Catalyst 800 instruments 1 800 831 6844 then press 24 1 650 638 5981 ABI PRISM 3100 Genetic Analyzer 1 800 831 6844 then press 26 1 650 638 5981 Biolnformatics includes BioLIMS BioMerge and SQL GT applications 1 800 831 6844 then press 25 1 505 982 7690 Peptide Synthesis 433 and 43X Systems 1 800 831 6844 then press 31 1 650 638 5981 Protein Sequencing Procise Protein Sequencing Systems 1 800 831 6844 then press 32 1 650 638 5981 PCR and Sequence Detec
63. 0 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Function 148 131 53 51 148 61 148 131 111 137 140 26 30 136 131 53 51 148 61 148 131 140 26 30 136 131 53 51 148 61 148 131 140 26 30 136 131 53 51 148 61 Function Name Cartridge Wait Dry Cart top Del 82 Cart sensor Del 82 Cart top Cartridge Wait Del S3 Cart top Cartridge Wait Dry Cart top Wash Input Block S3 Flush Input Block Flush Large Loop Cart Load R3 Cart lg loop Transfer R3 Cart gas Flush Cart Solvent Block Dry Cart top Del S2 Cart sensor Del S2 Cart top Cartridge Wait Del S3 Cart top Cartridge Wait Dry Cart top Flush Large Loop Cart Load R3 Cart Ig loop Transfer R3 Cart gas Flush Cart Solvent Block Dry Cart top Del S2 Cart sensor Del S2 Cart top Cartridge Wait Del S3 Cart top Cartridge Wait Dry Cart top Flush Large Loop Cart Load R3 Cart lg loop Transfer R3 Cart gas Flush Cart Solvent Block Dry Cart top Del S2 Cart sensor Del S2 Cart top Cartridge Wait Del S3 Cart top Cycle Method and Gradient Listings Time sec 5 30 20 10 5 10 5 60 10 30 10 50 5 15 30 20 10 5 10 5 30 10 50 5 15 30 20 10 5 10 5 30 10 50 5 15 30 20 10 5 10 Global Time N Z Z Z Z Z z Z Zz Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z
64. 03 218 340 213 215 118 121 222 Function Name Load X3 Cart Ig loop Flush Large Loop Cart Load X3 Cart sm loop Flush Small Loop Cart Flush Large Loop Cart Select Cartridge A Wait X3 to Cart A bottom Dry Cart bottom Select Cartridge B Wait Transfer to Flask X3 Transfer to Flask gas ET Empty Flask Select Cartridge C Wait Transfer to Flask X3 Dry Cart top Select Cartridge D Wait Transfer to Flask X3 Transfer to Flask gas End Cartridge Select Flush Cart Solvent Block Flush Output Block X3 to R5 Flush Large Loop Flask X3 to R4 Flush Large Loop Flask Load X3 Flask sm loop Flush Small Loop Flask X3 to X2g Backflush X2g Load X3 Flask Ig loop Flush Large Loop Flask X3 to S4 Dry Flask Empty Flask Transfer to Flask X3 Transfer to Flask gas Flush Flask Injector Global Time sec Time 60 5 60 30 60 60 60 10 60 30 10 30 10 10 N Z Z Zz Z Z Zz Z Zz Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Procedure Listings C 21 Step 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 Function 226 201 213 222 9 19 29 49 59 69 79 97 99 206 156 166 176 186 196 259 System Flush Total run time 75 00 Argon C 22 Procedure Listings Step AN DU FWD 11 12 13 14 15 16 Function 258 303 305 303 305 303 305 303 305 303
65. 13 23 17 18 19 20 34 35 36 37 40 13 10 17 18 19 20 34 35 36 37 16 13 23 17 18 19 20 34 35 36 37 40 13 23 16 7 11 13 22 7 11 13 21 52 52 13 52 15 5 7 17 18 19 20 34 35 36 37 40 5 11 10 17 18 19 20 34 35 36 37 16 5 7 17 18 19 20 34 35 36 37 40 5 1 5 7 22 5 7 21 59 59 Global Value 0 o O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O N o Sensor Function N ZEKXKXxXZ lt xZ ZZ Z Z Z Kox Z YxZ ZZ Z K Z lt Zx Z ZZ Z Z Z lt lt Z lt z Zz z z Function Listing Global Time Y lt Z lt lt Z lt lt lt Z Z lt Z lt lt Z lt lt Z Z Z lt Z lt lt Z lt lt lt Z Z lt Z lt lt Z lt lt lt Z Z A 3 Number 079 080 081 082 083 084 085 086 087 088 089 090 091 092 093 094 095 096 097 098 099 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 A 4 Function Listing Reagent XI X1 X1g X1g X1g X1g X1g X1g X1g X1g X1g X1g X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 S1 S1 S1 S1 S1 S2 S2 S2 S2 S2 S3 S3 S3 S3 S3 X3 X3 X3 Name Backflush X1 Reserved Del X1g Cart top Del X1g Cart bottom Not Available Del X1g Waste Not Available Not Available Vent X1g Flush X1g Backflush X1g Reserved Del X3 Cart top Del X3 Cart bottom Del X3 Cart sensor Del X3 Waste Load X3 C
66. 19 307 Compare Pressures 10th psi 0 N 20 309 Restore Reg Setpoint 0 N 21 285 Injector Sim Load 10 N 22 259 End 0 N Cart Reagent BIK Total run time 1 20 Test Global Step Function 4 Function Name Time sec Time 1 258 Begin 0 N 2 303 Select Regulator 5 N 3 304 Save Regulator Setpoint 0 N 4 131 Dry Cart top 5 N 5 305 Set Reg Setpoint 10th psi 38 N 6 310 Set Tolerance 100th psi 20 N 7 284 Open Valves 11 15 15 N 8 308 Close Pressure Valve 1 N Procedure Listings C 41 Step 10 11 12 13 14 15 16 17 18 19 20 Function 284 307 317 310 284 318 345 310 307 309 344 259 Cart Input BIk Test Total run time 1 20 C 42 Procedure Listings Step AN OOA 0 PM 11 12 13 14 15 16 17 18 19 20 Function 258 303 304 131 305 310 293 308 293 307 317 310 293 318 344 310 307 309 344 259 Function Name Open Valves 11 15 Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Open Valves 11 15 Compare Saved Pressure Open Valves 1 11 15 Set Tolerance 100th psi Compare Pressures 10th psi Restore Reg Setpoint Open Valves 7 11 15 16 End Function Name Begin Select Regulator Save Regulator Setpoint Dry Cart top Set Reg Setpoint 10th psi Set Tolerance 100th psi Open Valves 15 23 Close Pressure Valve Open Valves 15 23 Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Open Valves 15 23 Compare Saved Pre
67. 2 Bit A D Test 24 Bit A D Test Rheodyne End Global Time sec Time O MN PN PD PN INNO Z ZZ Z Zz Z Z Procedure Listings C 25 Flow Procedure List Flask Optimization Total run time 17 25 C 26 Procedure Listings Step MN O MAO PM 0 0 M M D M ND NON NN amp a a 44 so O O O N O O Asa 0 NP O O 000 N O Function 258 123 121 215 173 213 218 63 257 121 212 53 121 212 213 301 236 301 218 153 213 218 173 218 257 213 301 237 215 147 259 Function Name Begin Select Cartridge A Transfer to Flask gas Empty Flask Load SA Flask Ig loop Dry Flask Flush Large Loop Flask Del S3 Cart sensor Wait Transfer to Flask gas Bubble Flask Del S2 Cart sensor Transfer to Flask gas Bubble Flask Dry Flask Pause Pre Conversion Dry Pause Flush Large Loop Flask Load R4 Flask Ig loop Dry Flask Flush Large Loop Flask Load S4 Flask Ig loop Flush Large Loop Flask Wait Dry Flask Pause Post Conversion Dry Empty Flask End Cartridge Select End Time sec 0 0 30 15 15 10 10 15 0 45 40 15 45 40 120 100 10 15 10 10 15 10 60 300 100 15 Global Time Z 2 2 2 gt lt 2 2 2 Z Z 2 2 2 ZZZ ZZ 2 Z2 2 RZ ZZZ Z Zz Z Z Sensor amp Delivery Total run time 25 01 Test Step AN OA R 0 PM Q 0 0 0 0 0 0 0 0 83 NN PD IN D D M PD MMM SF aaa a Au O O N DOA BON O O N DO
68. 222 171 213 215 309 205 259 Function Name Backflush S4 Backflush X2 Backflush X2g Backflush X3 Flask End Function Name Begin Select Regulator Save Regulator Setpoint Set Reg Setpoint 10th psi Empty Flask Del X3 Flask Del X3 Waste Wash Input Block X3 Wash Output Block X3 Wait Flush Cart Solvent Block Flush Large Loop Cart Flush Cart Reagent Block Flush Input Block Flush Transfer Line Bubble Flask Dry Flask Empty Flask Del X3 Flask Flush Small Loop Flask Flush Large Loop Flask Dry Flask Load Position Injector Sim Load Flush Flask Injector Del 54 Flask Dry Flask Empty Flask Restore Reg Setpoint Flush X3 Flask End Time sec 20 60 20 60 0 Time sec 0 8 0 50 5 30 20 60 60 120 40 30 40 40 40 20 30 20 20 20 40 80 15 10 20 Global Time N ZZ ZZ Global Time Z ZIZ Z ZZ ZZ Z ZZ ZZ ZEN Ri 2 2 Z ZZ Z ZZ IZ Startup Procedure Startup Total run time 14 15 Step AN Oa Asa 0 PM WU 0 0 OOO 0 NP PN N PD PD M MMM N a a 44 4 a 4 0 NO TOF N O O UN DOA A 0 ON O 0 O JO O BR O N O Function 258 8 4 135 78 74 135 18 14 58 54 136 143 135 303 304 305 28 24 34 309 136 144 136 68 64 136 48 44 136 144 106 111 107 112 137 138 Function Name Begin Flush R1 Del R1 Waste Flush Cart Reagent Block Flush X1 Del X1 Waste Flush Cart Reagent Block Flush R2g Del R2g Waste
69. 257 41 42 41 42 41 42 41 42 41 148 41 148 42 147 135 136 137 138 139 140 123 131 147 124 131 147 125 131 147 126 131 147 Function Name Del S1 Cart bottom Del S1 Cart top Cartridge Wait Del S1 Cart top Cartridge Wait Del S1 Cart bottom End Cartridge Select Select Cartridge D Wait Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart Del S1 Cart top Del S1 Cart bottom Del S1 Cart top Cartridge Wait Del S1 Cart top Cartridge Wait Del S1 Cart bottom End Cartridge Select Flush Cart Reagent Block Flush Cart Solvent Block Flush Input Block Flush Output Block Flush Small Loop Cart Flush Large Loop Cart Select Cartridge A Dry Cart top End Cartridge Select Select Cartridge B Dry Cart top End Cartridge Select Select Cartridge C Dry Cart top End Cartridge Select Select Cartridge D Dry Cart top End Cartridge Select top bottom top bottom top bottom ni ning ms ERI NE ee EI Global Time sec Time 10 60 30 60 30 60 0 0 120 120 10 10 10 10 10 10 10 60 30 60 30 60 90 90 90 90 90 90 90 90 90 N Z Z Zz Z Z z Z Zz Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Procedure Listings C 19 Step 87 Function 259 System Clean Out Total run time 26 44 X3 C 20 Procedure Listings Step O N Oa R 0 PM KM 0 0 0 0 82 NP PD N NNN M MMM SF o
70. 3 24 Function 218 259 Bottle Change for R5 Total run time 1 25 Step AN Oa FWD 11 12 Function 258 265 303 164 166 260 165 167 218 220 218 259 Bottle Change for Total run time 2 50 R5 Leak Step O N DOA R 0 PM 11 12 13 14 15 16 17 Function 258 265 303 304 164 166 260 305 310 257 308 257 307 317 310 257 318 Function Name Flush Large Loop Flask End Function Name Begin Set for Bottle R5 Select Regulator Vent R5 Backflush R5 Pause for Bottle Change Flush R5 Del R5 Waste Flush Large Loop Flask Wash Large Loop Flask Flush Large Loop Flask End Function Name Begin Set for Bottle R5 Select Regulator Save Regulator Setpoint Vent R5 Backflush R5 Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Global Time sec Time 20 0 N N Global Time sec Time 0 0 7 10 15 0 10 10 10 10 20 0 N Z Z Z Z Z Z Z Z Z Z Z Global Time sec Time 0 0 7 0 10 15 0 38 20 40 0 15 38 30 N Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Procedure Listings C 7 Bottle Change for S1 Step 18 19 20 21 22 23 24 Function 309 165 167 218 220 218 259 Total run time 1 30 Step O J O OAO PM gt A N O Functi
71. 3 Waste Flush Cart Reagent Block Wash Cart Reagent Block Flush Cart Reagent Block Del X3 Waste Flask Flush Large Loop Flask End Function Name Begin Select Regulator Save Regulator Setpoint Bubble Flask Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Bubble Flask Set Tolerance 100th psi Compare Pressures 10th psi Restore Reg Setpoint Bubble Flask Time sec 0 5 30 0 40 10 60 10 10 30 20 10 Time sec 0 9 0 5 38 20 25 1 15 38 0 10 30 0 15 10 0 0 5 Global Time N ZZ A Z Zz Z Z Z ZZ ZZ ZZ Global Time N 2 2 2 Z Z ZZ Z ZZ Z Z Z ZZ Z ZZ Global Step Function Function Name Time sec Time 20 259 End 0 N Regulator 10 Test Total run time 1 55 Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 303 Select Regulator 10 N 3 304 Save Regulator Setpoint 0 N 4 223 Inject Position 0 N 5 257 Wait 5 N 6 221 Flush Injector 10 N 7 305 Set Reg Setpoint 10th psi 38 N 8 310 Set Tolerance 100th psi 20 N 9 257 Wait 25 N 10 308 Close Pressure Valve 1 N 11 257 Wait 15 N 12 307 Compare Pressures 10th psi 38 N 13 317 Save Regulator Pressure 0 N 14 310 Set Tolerance 100th psi 10 N 15 257 Wait 30 N 16 318 Compare Saved Pressure 0 N 17 285 Injector Sim Load 20 N 18 310 Set Tolerance 100th psi 10 N
72. 3 18 Flush R2g 10 N 4 28 Flush R3 5 N 5 48 Flush S1 10 N 6 58 Flush S2 15 N 7 68 Flush S3 10 N 8 78 Flush X1 5 N 9 98 Flush X3 Cart 10 N 10 155 Flush R4 5 N 11 165 Flush R5 3 N 12 175 Flush S4 10 N 13 185 Flush X2 5 N 14 259 End 0 N Procedure Listings C 29 Init Sensor Procedure Init Sensor Total run time 9 16 Procedure C 30 Procedure Listings Step MN O MAO PM U U G KM G GQ M M M M M M M MMM SF a aaa a Ada Oo S SZ RZ BS SE amp N D D D N D do a RE D Z Function 258 145 139 273 146 140 274 107 112 138 123 132 275 147 124 132 276 147 125 132 277 147 126 132 278 147 149 141 279 215 219 217 280 220 218 281 226 Function Name Begin Wash Small Loop Cart Flush Small Loop Cart Init Sm Loop Snsr Cart Wash Large Loop Cart Flush Large Loop Cart Init Lg Loop Snsr Cart Wash Output Block S2 Wash Output Block S3 Flush Output Block Select Cartridge A Dry Cart bottom Init Cart A Snsr End Cartridge Select Select Cartridge B Dry Cart bottom Init Cart B Snsr End Cartridge Select Select Cartridge C Dry Cart bottom Init Cart C Snsr End Cartridge Select Select Cartridge D Dry Cart bottom Init Cart D Snsr End Cartridge Select Wash Transfer Line S2 Flush Transfer Line Init Transfer Snsr Empty Flask Wash Small Loop Flask Flush Small Loop Flask Init Sm Loop Snsr Flask Wash Large Loop Flask Flush Large Loop Flask
73. 30 30 10 40 15 10 10 10 10 30 10 Time sec 0 30 Global Time N Z Zz Zizi ZZZ ZZ Z Z ZZZ z2 2 Z 2 2 Z 2 2 2 Z x Z Z Z Z Z Global Time N N Global Step Function Function Name Time sec Time 3 227 Prepare Pump 1 N 4 257 Wait 480 N 5 232 Start Gradient 1 N 6 257 Wait 690 N 7 257 Wait 690 N 8 259 End 0 N Flask Prep Pump Total run time 1 21 Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 257 Wait 30 N 3 227 Prepare Pump 1 N 4 257 Wait 50 N 5 259 End 0 N Injector Optimize Total run time 4 36 Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 235 Set as Residue Cycle 0 N 3 218 Flush Large Loop Flask 10 N 4 215 Empty Flask 20 N 5 226 Load Position 1 N 6 173 Load S4 Flask lg loop 15 N 7 213 Dry Flask 10 N 8 218 Flush Large Loop Flask 10 N 9 173 Load S4 Flask lg loop 15 N 10 213 Dry Flask 10 N 11 218 Flush Large Loop Flask 10 N 12 221 Flush Injector 30 N 13 257 Wait 5 N 14 221 Flush Injector 30 N 15 213 Dry Flask 5 N 16 238 Concentrate Sample 0 Y 17 257 Wait 10 N 18 225 Load Injector 40 N 19 249 Inject Pos Collect Data 1 N 20 222 Flush Flask Injector 30 N 21 221 Flush Injector 10 N Cycle Method and Gradient Listings B 29 Step 22 Function 259 Manual Injection Total run time 31 33 Step O ON OOO Aa ON Function 258 226 257 227 257 249 232 257 257 259 Flask Optimize Flsk Total run time 19 31 Step
74. 31 Dry Cart top 40 N 3 127 Ready Transfer to Flask 0 N 4 141 Flush Transfer Line 5 N 5 63 Del S3 Cart sensor 15 N 6 148 Cartridge Wait 10 N 7 121 Transfer to Flask gas 45 N 8 141 Flush Transfer Line 5 N 9 53 Del S2 Cart Sensor 15 N Cycle Method and Gradient Listings B 21 Step 10 11 12 13 14 15 16 17 18 Function 148 121 141 63 148 121 128 131 259 Cart PL Proline Total run time 33 45 Step O N o asa 0 PM M PO N N N N N N a H A A BH da S CE ND OF 0 N O O O N N DOA ON O B 22 Cycle Method and Gradient Listings Function 258 137 11 140 6 131 140 135 11 132 140 6 131 140 135 11 132 140 6 131 140 135 11 146 111 140 137 Function Name Cartridge Wait Transfer to Flask gas Flush Transfer Line Del S3 Cart sensor Cartridge Wait Transfer to Flask gas Transfer Complete Dry Cart top End Function Name Begin Flush Input Block Del R2g Cart top Flush Large Loop Cart Load R1 Cart Ig loop Dry Cart top Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Dry Cart bottom Flush Large Loop Cart Load R1 Cart Ig loop Dry Cart top Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Dry Cart bottom Flush Large Loop Cart Load R1 Cart Ig loop Dry Cart top Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Wash Large Loop Cart Wash Input Block S3
75. 4 leak 2 40 Bottle Change for X1 3 25 Bottle Change for X1 leak 4 50 Bottle Change for X2 2 05 Bottle Change for X2 leak 3 30 Bottle Change for X3 both 5 45 Bottle Change for X3 both leak 7 10 Changing a Bottle IMPORTANT Leak tests use functions that alter the pressure management system pressure setting If the procedure is aborted before the end step select the Pressure amp Temperature view and click on the Default button to restore the operating pressures To change a bottle Step Action 1 Select the Bottle Change View from the View pop up menu Do not remove the bottle at this time Highlight the chemical to be changed 3 Enter the lot number of the chemical in the Lot Number box The date will be updated automatically Click on the Change Bottle button 5 a When prompted remove the old bottle and bottle seal b Put a new bottle seal on the new bottle rim c Screw the bottle into the bottle cap assembly 6 Tighten until the bottle seal contacts the top of the bottle cap assembly and then turn approximately a quarter turn more IMPORTANT It is not necessary to tighten a bottle until a snapping sound ratcheting is produced by the bottle cap assembly Ratcheting the bottle cap assembly will cause premature wear and may crack the bottle seal 7 Click on the Continue button The bottle change procedure will continue through the remaining steps which includes priming the
76. 4 15 16 17 18 19 20 21 22 23 24 25 Function 258 271 303 304 184 186 196 260 305 310 257 308 257 307 317 310 257 318 309 185 187 197 220 218 259 Bottle Change for X3 Total run time 5 45 Both C 14 Procedure Listings Step 1 2 Function 258 272 Function Name Del X2g Waste Wash Large Loop Flask Flush Large Loop Flask End Function Name Begin Set for Bottle X2 Select Regulator Save Regulator Setpoint Vent X2 Backflush X2 Backflush X2g Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Restore Reg Setpoint Flush X2 Del X2 Waste Del X2g Waste Wash Large Loop Flask Flush Large Loop Flask End Function Name Begin Set for Bottle X3 Time sec 10 10 20 0 Time sec 0 0 7 0 10 20 20 0 38 20 40 0 15 38 0 5 30 0 0 15 20 10 10 20 0 Time sec 0 0 Global Time N N N N Global Time N Z Z Ze ZZ Zz ZZ i n rr ZZ Global Time Step aN DOO As 0 11 12 13 14 15 16 17 Function 303 97 99 206 260 98 94 135 143 135 207 218 220 218 259 Bottle Change for X3 Total run time 7 10 Both Leak Step AN DOA Asa 0 PM ND N O O O N O G Asa 0 MN O Function 258 272 303 304
77. 6 off Cart dry 3 5 psi Valve 46 on All cart block flushing Internal manual regulator pressure Valve 47 off Flask dry all flask flushing 3 0 psi Valve 47 on Load injector 1 0 psi Valve 48 off Flask bubble low pressure sample loop 1 8 psi flushing Valve 48 on High pressure sample loop flushing Internal manual regulator pressure Valves Functions Cycles and Methods 3 5 Functions About Functions Function Numbering Format Valve Controlling Functions Sensor Functions Cycle Synchronizing Functions For the instrument to perform any task such as delivering a reagent valves must be activated To simplify programming and operation valves are grouped into functions Each function is numbered and given a name that describes its purpose Table 3 2 provides an outline of the function numbering and naming format The complete function list can be found in Appendix A Function Listing or from the Functions view in the Procise software The function numbering system is Standard functions are numbered 1 400 User functions are numbered 401 450 Table 3 2 Function Numbering Format Function Number Function 1 150 Cartridge 151 250 Flask HPLC 251 259 Transfer 260 360 Test Procedure 401 450 User These functions activate a valve or set of valves simultaneously to deliver a chemical or gas These functions are also called time dependent functions because the valves are ac
78. 7 048 049 050 051 052 053 054 055 056 057 058 059 060 061 062 063 064 065 066 067 068 069 070 071 072 073 074 075 076 077 078 Reagent R3g R3g R3g S1 S1 S1 S1 S1 S1 S1 S1 S1 S1 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S3 S3 S3 S3 S3 S3 S3 S3 S3 S3 X1 X1 X1 X1 X1 X1 X1 X1 Name Flush R3g Backflush R3g Reserved Del S1 Cart top Del S1 Cart bottom Del S1 Cart sensor Del S1 Waste Load S1 Cart sm loop Load S1 Cart Ig loop Vent S1 Flush S1 Backflush S1 Reserved Del S2 Cart top Del S2 Cart bottom Del S2 Cart sensor Del S2 Waste Load S2 Cart sm loop Load S2 Cart Ig loop Vent S2 Flush S2 Backflush S2 Reserved Del S3 Cart top Del S3 Cart bottom Del S3 Cart sensor Del S3 Waste Load S3 Cart sm loop Load S3 Cart Ig loop Vent S3 Flush S3 Backflush S3 Reserved Del X1 Cart top Del X1 Cart bottom Del X1 Cart sensor Del X1 Waste Load X1 Cart sm loop Load X1 Cart Ig loop Vent X1 Flush X1 Valves 53 9 53 15 14 23 17 18 19 20 34 35 36 37 40 14 10 17 18 19 20 34 35 36 37 16 14 23 17 18 19 20 34 35 36 37 40 14 23 16 7 11 14 22 7 11 14 21 56 56 14 56 15 12 23 17 18 19 20 34 35 36 37 40 12 10 17 18 19 20 34 35 36 37 16 12 23 17 18 19 20 34 35 36 37 40 12 23 16 7 11 12 22 7 11 12 21 54 54 12 54 15
79. 9 Restore Reg Setpoint N 32 185 Flush X2 5 N 33 74 Del X1 Waste 70 N 34 135 Flush Cart Reagent Block 10 N 35 143 Wash Cart Reagent Block 10 N 36 135 Flush Cart Reagent Block 30 N 37 187 Del X2 Waste 15 N 38 218 Flush Large Loop Flask 10 N 39 167 Del R5 Waste 5 N 40 218 Flush Large Loop Flask 10 N 41 220 Wash Large Loop Flask 10 N 42 218 Flush Large Loop Flask 30 N 43 259 End 0 N X3 Leak Test Total run time 5 15 Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 303 Select Regulator 8 N 3 304 Save Regulator Setpoint 0 N 4 97 Vent X3 Cart 5 N 5 99 Backflush X3 Cart 20 N 6 206 Backflush X3 Flask 20 N 7 305 Set Reg Setpoint 10th psi 38 N 8 310 Set Tolerance 100th psi 20 N 9 257 Wait 40 N 10 308 Close Pressure Valve 1 N 11 257 Wait 15 N 12 307 Compare Pressures 10th psi 38 N Procedure Listings C 39 Step 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Function 317 310 257 318 97 310 307 309 98 94 135 143 135 207 218 259 Regulator 9 Test Total run time 1 35 C 40 Procedure Listings Step AN Oa FWD 11 12 13 14 15 16 17 18 19 Function 258 303 304 212 305 310 257 308 257 307 317 310 257 318 212 310 307 309 212 Function Name Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Vent X3 Cart Set Tolerance 100th psi Compare Pressures 10th psi Restore Reg Setpoint Flush X3 Cart Del X
80. A E ON O DAN DOA BR GM Function 258 226 139 5 139 143 145 135 139 140 76 140 135 303 304 305 26 309 34 136 140 144 143 111 107 146 135 137 138 140 136 123 43 131 53 131 63 131 118 Function Name Begin Load Position Flush Small Loop Cart Load R1 Cart sm loop Flush Small Loop Cart Wash Cart Reagent Block Wash Small Loop Cart Flush Cart Reagent Block Flush Small Loop Cart Flush Large Loop Cart Load X1 Cart Ig loop Flush Large Loop Cart Flush Cart Reagent Block Select Regulator Save Regulator Setpoint Set Reg Setpoint 10th psi Load R3 Cart Ig loop Restore Reg Setpoint Del R3g Waste Flush Cart Solvent Block Flush Large Loop Cart Wash Cart Solvent Block Wash Cart Reagent Block Wash Input Block S3 Wash Output Block S2 Wash Large Loop Cart Flush Cart Reagent Block Flush Input Block Flush Output Block Flush Large Loop Cart Flush Cart Solvent Block Select Cartridge A Del S1 Cart sensor Dry Cart top Del 82 Cart Dry Cart top sensor Del S3 Cart sensor Dry Cart top Transfer to Flask X3 Global Time sec Time 0 1 10 18 10 10 10 20 20 10 65 20 20 3 0 15 45 0 15 10 10 10 15 10 10 10 60 60 60 30 60 4 13 60 13 60 13 60 50 Z Z Z z Z z Z Zz Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z 2 Procedure Listings C 27 C 28 Procedure Listings Step 40 41 42 43
81. C in a dark dry place Supporting Supporting solvents and chemicals needed to run the system are listed below Chemicals Part Storage Chemicals Number Conditions Solvent A3 3 5 THF Tetrahydrofuran in Water 1 L 401464 RT Procise Solvent B2 12 Isopropanol and Acetonitrile 1 L 401570 RT Procise Solvent B not used 100 acetonitrile 1 L 400313 RT Acetone HPLC grade RT MeOH Methanol 400470 RT Premix Buffer Concentrate 401446 4 C 1 6 Introduction and Safety Chemical Warnings Table Table 1 2 Warnings on Chemicals Used in the Procise System Chemical Chemical Hazard A3 3 5 tetrahydrofuran in water CHEMICAL HAZARD A3 tetrahydrofuran in water is a flammable liquid and vapor It may be harmful if swallowed Exposure may cause eye and respiratory tract irritation central nervous system depression and liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Acetone CHEMICAL HAZARD Acetone is a flammable liquid and vapor lt may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Acetonitrile Also in R5 S4B B B2
82. Flow Meter SCCM Tare Sartorius Log Weight X3 to R1 4 6 55 X3 to R3 4 8 11 53 X3 to R4 26 28 51 X3 to R5 26 29 49 X3 to S1 4 11 14 56 X3 to S2 4 11 12 54 X3 to S3 4 11 13 52 A 10 Function Listing Global Value o O O O O O O O O O O O O O O O O O O Sensor Function N N N Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Global Time N N Z Z ZZ Z ZZ Z 2222 Z ZIZ Z Z Z Z Z Z Z Number 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 400 401 450 Reagent Name X3 to S4 X3 to X1 X3 to X2 X3 to Cart A bottom Open Valves 7 11 15 16 Open Valves 1 11 15 Open Valves 1 11 Open Valves 1 15 16 40 Open Valves 11 15 16 Open Valves 15 23 Open Valves 10 15 45 Open Valve 30 Flask Reag BIk Test Open Valves 24 45 Open Valve 43 Open Valves 44 45 Reserved User Functions 1 50 Valves 25 26 50 4 5 59 26 27 57 4 10 11 16 17 34 7 11 15 16 1 11 15 1 11 1 15 16 40 11 15 16 15 23 10 15 45 Global Value 0 o O O O O O O O O O O O O O O O Sensor Function N Z Z ZZ ZZ Z ZZ Z Z sl Z Z Z Z Z Function Listing Global Time N Z 2 Z 2 202Z Z Z Z Z Z Z Z Z Z Z Z A 11 Cycle Method and Gradient Listings Overview About This Tables of cycles methods and gradients are in this appendix Appendix In This Appendix This appendix contains the follo
83. Flush S2 Del S2 Waste Flush Cart Solvent Block Wash Cart Reagent Block Flush Cart Reagent Block Select Regulator Save Regulator Setpoint Set Reg Setpoint 10th psi Flush R3 Del R3 Waste Del R3g Waste Restore Reg Setpoint Flush Cart Solvent Block Wash Cart Solvent Block Flush Cart Solvent Block Flush S3 Del S3 Waste Flush Cart Solvent Block Flush S1 Del S1 Waste Flush Cart Solvent Block Wash Cart Solvent Block Wash Input Block S2 Wash Input Block S3 Wash Output Block S2 Wash Output Block S3 Flush Input Block Flush Output Block Time sec 0 10 15 10 15 60 10 10 10 15 15 20 15 20 3 0 15 10 60 10 0 10 15 20 15 15 10 15 10 10 15 15 15 15 15 40 40 Global Time Z Z Z z Z Z Z Z Z Zz Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Procedure Listings C 47 C 48 Procedure Listings Step 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Function 136 155 157 218 165 167 218 175 177 218 171 213 215 171 213 215 259 Function Name Flush Cart Solvent Block Flush R4 Del R4 Waste Flush Large Loop Flask Flush R5 Del R5 Waste Flush Large Loop Flask Flush S4 Del S4 Waste Flush Large Loop Flask Del 54 Flask Dry Flask Empty Flask Del S4 Flask Dry Flask Empty Flask End Time sec 40 10 30 10 5 10 10 15 15 20 15 10 20 15 10 40 0 Global Time N Z ZZ Z Z ZZ Z ZIZ Z Z Zz ZZ G
84. Function Name Time sec Time 1 258 Begin 0 N 2 139 Flush Small Loop Cart 10 N 3 75 Load X1 Cart sm loop 70 Y 4 131 Dry Cart top 30 N 5 139 Flush Small Loop Cart 10 N 6 135 Flush Cart Reagent Block 5 N 7 11 Del R2g Cart top 30 N 8 140 Flush Large Loop Cart 10 N 9 6 Load R1 Cart Ig loop 20 N 10 131 Dry Cart top 30 N 11 140 Flush Large Loop Cart 5 N 12 135 Flush Cart Reagent Block 5 N 13 11 Del R2g Cart top 170 N 14 132 Dry Cart bottom 30 N 15 140 Flush Large Loop Cart 5 N 16 6 Load R1 Cart Ig loop 20 N 17 131 Dry Cart top 30 N 18 140 Flush Large Loop Cart 5 N 19 135 Flush Cart Reagent Block 5 N 20 11 Del R2g Cart top 170 N 21 132 Dry Cart bottom 30 N 22 140 Flush Large Loop Cart 5 N Cycle Method and Gradient Listings B 15 Step 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 B 16 Cycle Method and Gradient Listings Function 6 131 140 135 11 146 111 140 137 136 132 142 63 148 61 148 51 148 51 148 131 53 148 51 148 51 148 61 148 131 34 31 143 135 144 136 131 142 127 141 63 Function Name Load R1 Cart Ig loop Dry Cart top Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Wash Large Loop Cart Wash Input Block S3 Flush Large Loop Cart Flush Input Block Flush Cart Solvent Block Dry Cart bottom Set Car
85. Glu needs to be separated from DMPTU increase the initial B If the Asp DTT peak separation will not be compromised increase the initial B by 2 3 add a gradient step at 0 4 minutes If increasing the initial B will cause the Asp to elute too early add a gradient step at 0 4 minutes and set the B to 14 16 Improving the To improve the Then Separation of Other A Met Val separation increase the column oven temperature Amino Acids p 4 Increase the temperature in 2 C increments Do not raise the temperature above 59 C lle Lys separation decrease the B at 18 minutes If the peaks are more than 50 separated decrease the B by 1 If the peaks are less than 50 separated decrease the B by 2 Lys Leu separation increase the B at 18 minutes If the peaks are more than 50 separated increase the B by 1 If the peaks are less than 50 separated increase the B by 2 7 10 PTH Separation Optimization Table In Table 7 3 the arrow above an amino acid indicates the direction in which the peak moves after changing the variable listed in the column Left is toward the injection point Table 7 3 Optimization Guidelines Variable Major Effect Minor Effect Increase initial B E E ES DTT D PTU E DMPIU H SR Decrease initial B gt gt E sy 3 DTT D PTU S Q E DMPTU H Increase initial B at 0 4 e E He E DMPTU H S Ri S
86. I press Flush Small Loop Flask Flush Large Loop Flask Wash Small Loop Flask Wash Large Loop Flask Flush Injector Flush Flask Injector Inject Position Flush Injector Low Pres Load Injector Load Position Prepare Pump Ready to Receive Valves 57 57 33 57 24 31 33 26 32 45 26 30 26 31 60 60 26 60 24 26 31 41 44 45 48 41 44 45 24 32 41 44 45 24 32 41 44 45 48 24 32 41 43 24 32 41 43 47 24 30 24 31 25 30 25 31 42 44 48 24 32 41 42 42 44 24 32 41 42 47 41 44 45 Global Value 0 o O O O O O O O O O O O O O O O O O O O O O O O o O O O o O O Sensor Function N E ZZ Z Z Z Z Z ZZ E Z Z Z Z 2 Z Z Z Z x ye ae Z Z Z zZz x Global Time N lt lt Z Z Z Z lt lt lt lt lt lt lt lt lt lt Z Z Z Z lt lt lt lt Z lt Z lt lt lt lt lt lt lt lt Function Listing A 7 Global Sensor Global Number Reagent Name Valves Value Function Time 229 Set Column 0 N Y Temperature 230 Set Flask Temperature 0 N Y 231 Stop Pump 0 N Y 232 Start Gradient 0 N N 233 Set as Blank Cycle 0 N N 234 Set as Standard Cycle 0 N N 235 Set as Residue Cycle 0 N N 236 Pre Conversion Dry 2 4 32 41 44 45 80 N Y 237 Post Conversion Dry 24 32 41 44 45 200 N Y 238 Concentrate Sample 24 32 41 44 45 15 N Y 239 Reserved 0 N N 240 Reserved 0 N N 241 Reserved 0 N
87. ITC to create a phenylthiocarbamyl PTC protein peptide It includes Delivery of PITC Delivery of base vapor to provide the basic environment necessary for coupling Drying and washing to remove excess reagent and reaction by products The coupling reaction used for samples bound to PVDF membrane is slightly different from the coupling for samples applied to glass fiber Sequencing cycles typically have been written for samples applied to a hydrophilic support The hydrophilicity of the support facilitates the absorption of a small amount of water necessary for efficient coupling of PITC to the amino terminus of the sample PVDF membrane is routinely used for electroblotting of samples from gels and can be used for the clean up of salt and buffer containing samples prior to loading on the protein sequencer PVDF membrane binds proteins through hydrophobic interaction Because PVDF membrane is hydrophobic its tendency is to repel water rather than absorb it In the coupling reaction of the blot cycles an aliquot of 50 methanol in water is delivered from the X1 bottle position to the reaction cartridge in order to wet the membrane prior to coupling A brief argon drying step removes the methanol leaving the sample solvated with a small amount of water when the coupling reaction begins This addition improves the coupling efficiency for PVDF membrane bound samples All the standard chemistry cycles use three deliveries of PITC during the co
88. Init Lg Loop Snsr Flask Load Position Time sec 0 10 30 0 10 30 0 15 15 40 0 30 40 60 10 30 10 30 Global z g 3 o Zz Z ZE z Z ZZ Z Z Zz Z zz z Z Z Liu 2 ZZ zz Zz Zz ZZ Z ZZ Z Z Step 39 40 41 42 43 44 Function 221 257 221 282 283 259 Function Name Flush Injector Wait Flush Injector Init Injector Load Snsr Init Injector Full Snsr End Time sec 30 5 30 Procedure Listings Global Time N Z Z Z Z Z C 31 Leak Procedure List Cartridge A Leak Total run time 1 36 Test Step MN o asa WD N a LO 0 JO dl 8 ON Function 258 303 304 123 131 257 305 310 129 308 129 307 317 310 129 318 309 131 147 259 Cartridge B Leak Total run time 1 36 Test Step AN O fF WD gt A N a o C 32 Procedure Listings Function 258 303 304 124 131 257 305 310 129 308 129 307 Function Name Begin Select Regulator Save Regulator Setpoint Select Cartridge A Dry Cart top Wait Set Reg Setpoint 10th psi Set Tolerance 100th psi Pressurize Cart top Close Pressure Valve Pressurize Cart top Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Pressurize Cart top Compare Saved Pressure Restore Reg Setpoint Dry Cart top End Cartridge Select End Function Name Begin Select Regulator Save Regulator Setpoint Select Cartridge B Dry Cart
89. N 242 Reserved 0 N N 243 Reserved 0 N N 244 Reserved 0 N N 245 Reserved 0 N N 246 Reserved 0 N N 247 Reserved 0 N N 248 Reserved 0 N N 249 Reserved 0 N N 250 Reserved 0 N N 251 490A Relay 1 Off 0 N Y 252 490A Relay 1 On 0 N Y 253 490A Relay 1 Pulse 0 N Y 254 490A Relay 2 Off 0 N Y 255 490A Relay 2 On 0 N Y 256 490A Relay 2 Pulse 0 N Y 257 Wait 0 N Y 258 Begin 0 N N 259 End 0 N N 260 Pause for Bottle 0 N N Change 261 Set for Bottle R1 0 N N 262 Set for Bottle R2 0 N N 263 Set for Bottle R3 0 N N 264 Set for Bottle R4 0 N N 265 Set for Bottle R5 0 N N 266 Set for Bottle S1 0 N N 267 Set for Bottle S2 0 N N 268 Set for Bottle S3 0 N N A 8 Function Listing Number 269 270 271 272 273 274 275 276 277 278 279 280 Reagent 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 Name Set for Bottle S4 Set for Bottle X1 Set for Bottle X2 Set for Bottle X3 Init Sm Loop Snsr Cart Init Lg Loop Snsr Cart Init Cart A Snsr Init Cart B Snsr Init Cart C Snsr Init Cart D Snsr Init Transfer Snsr Init Sm Loop Snsr Flask Init Lg Loop Snsr Flask Init Injector Load Snsr Init Injector Full Snsr Open Valves Injector Sim Load X3 to R2 X3 to R3g X3 to X1g X3 to X2g Vent 16 Test Vent 30 Test Vent 43 Test Open Valves 15 23 Open Valve 24 Open Valves 24 32 Open Valves 24 32 45 Flask Out Test Flask Reag BIk Hi Test Op
90. Outlet A liquid sensor at the outlet of each cartridge simplifies optimization of the delivery of Sensors solvent to the cartridges for both washing and extraction The sensor eliminates the need for timing the delivery of solvent to the midpoint of the cartridge All washes and extractions in the standard cycles except for the wash after cleavage are controlled by these sensors Washings are controlled by a first delivery to the cartridge outlet sensor followed by short pulses of solvent alternated with wait steps Extractions are deliveries to the cartridge outlet sensor followed by a brief incubation and transfer to the flask Determining Deliver Note Whenever the delivery pressure for a reagent is changed load times must be changed to Cartridge 25 well Sensor Function IMPORTANT Ifthe protein sequencer has not been run since the last cold start use the Test Time view to select and run the Init Sensor procedure Then allow the procedure to run to completion To determine the amount of time necessary for a Deliver to Cartridge sensor function Step Action 1 From the Pressures and Temperatures view set the desired delivery pressure for the bottle position to be used 2 If the reagent or solvent is not loaded on the instrument perform the bottle change procedure for that bottle position 3 From the Manual Control view a Select Function 131 Dry Cart top from the cartridge function list b Activate the fun
91. Passage between a manifold inlet line and the manifold occurs only when the appropriate valve is activated The manifold zigzags through the valve block and passes other closed valves that are unaffected by the flow The direction of the flow is determined by the pressures on either side of the pathway See Figure 3 1 for the valve diagram on reagent solvent and gas flow Valves Functions Cycles and Methods 3 3 Cartridge Reagent Block Cartridge Solvent Block Flask Reagent Block O O O 1 2 3 4 5 6 7 8 9 10 11 12 13 Les 15 24 25 26 27 28 29 50 0 0 7 Q o 0 CAVE xl x 11 a R2 X3 FEN R3 R3 2 3 si E 6 a 4 RS V as liq ligji lig Y V liq gas T Uig ig lig lig ji iq iq lig ie W W h r N Com NO Flask Input Block Cartridge Input Block Argon Argon 31 32 33 Argon Argon Low High Om Q O High Low 16 17 18 19 20 21 22 23 g Ho OO OO OO Y as 6 Ka 45 W ri v yy We Wea WN Flask Output Block igicip L SiS 41 42 43 44 ii i E pp Tw Tie Ta Tn V FIC W n YY V J v y 48 Com 7 NC SNO O O OO OO Flask 34 35 36 37 38 39 40 O OSB O Argon Argon High L Cartridge Output Block 9 ica Injector Legend To Col
92. Procise Protein Sequencing System Advanced Operation User s Manual i Applied AS Biosystems Copyright 2000 2002 Applied Biosystems For Research Use Only Not for use in diagnostic procedures Applied Biosystems and PROCISE are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries ABI BioBrene Blott and ProSorb are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries All other trademarks are the sole property of their respective owners Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Contents Introduction and Safety OVEEVICW ivo VERTE ea a Pea 1 1 Sales A AE Dee ate OO AE 1 2 Reagents and Solvents Used on the System 1 6 Sequence Analysis Chemistry OVINA TET 2 1 TITO UC ION sjah s k ace A RE alia T 2 2 Edman Degrad tion ce a messe 2 3 Valves Functions Cycles and Methods Overview icaro aa nn nu de sud sn dd Senk SG TA Ge EU sk Raset 3 1 Components of a Run isse lire eb te ia 3 2 VALVES a een A A A TIR acne ke Es 3 3 FUNCHONS iure reader Se Vee SAGT fa ea B TE de OE EN 3 6 Cycles cristiana aa A Eek ane hse OU ee ead ab i 3 9 Methods 54h an A E hia aA skar 3 11 Creating Functions Cycles and Methods OVERVIEW in ae HE yh bows RANA
93. QT Decrease B at 0 4 minutes gt gt E DMPTU H Increase final B at 10 and 18 minutes IKL Decrease final B at 10 and gt 18 minutes IKL Increase column gt he temperature 2 C M V H R PECys Decrease column gt gt temperature 2 C S R TG Increase molarity HA R S PECys P Fast Gradients Only gt gt Decrease flow rate PTH Separation 7 11 Function Listing Overview About This This appendix lists all the function numbers from 1 450 and the valves and reagents Appendix associated with the function In This Appendix This appendix contains the following topic Topic See Page Function List Table A 2 Function Listing A 1 Function List Table Number 001 002 003 004 005 006 007 008 009 010 011 012 013 014 015 016 017 018 019 020 021 022 023 024 025 026 027 028 029 030 031 032 033 034 035 036 037 A 2 Function Listing Reagent RI RI RI RI RI RI RI RI RI RI R2g R2g R2g R2g R2g R2g R2g R2g R2g R2g R3 R3 R3 R3 R3 R3 R3 R3 R3 R3 R3g R3g R3g R3g R3g R3g R3g function Name Del R1 Cart top Del R1 Cart bottom Del R1 Cart sensor Del R1 Waste Load R1 Cart sm loop Load R1 Cart Ig loop Vent R1 Flush R1 Backflush R1 Reserved Del R2g Cart top Del R2g Cart bottom Not Available Del R2g Waste Not Available Not Available Vent R2g Flush R2g Backflush R2g Reser
94. RD R4 25 trifluoroacetic acid in water causes severe burns to the eyes skin and respiratory tract Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 1 8 Introduction and Safety Table 1 2 Warnings on Chemicals Used in the Procise System continued Chemical Chemical Hazard R5 0 001 DTT in acetonitrile CHEMICAL HAZARD R5 0 001 DTT in acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Sodium phosphate CHEMICAL HAZARD Sodium phosphate may cause eye skin and upper respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves S1 n heptane CHEMICAL HAZARD S1 n heptane is a flammable liquid and vapor lt may cause eye skin and respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness headache etc and irregular heartbeats Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves S2B ethyl acetate CHEMICAL HAZARD S2B ethyl acetate is a flam
95. Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Restore Reg Setpoint Flush S2 Del S2 Waste Flush Cart Solvent Block End Function Name Begin Set for Bottle S3 Select Regulator Vent S3 Backflush S3 Pause for Bottle Change Flush S3 Del S3 Waste Flush Cart Solvent Block End Function Name Begin Set for Bottle S3 Select Regulator Save Regulator Setpoint Vent S3 Backflush S3 Pause for Bottle Change Set Reg Setpoint 10th psi Time sec 40 0 15 38 30 15 10 20 Time sec 0 0 4 10 20 0 15 10 20 0 Time sec 0 0 4 0 10 20 0 38 Global Time N ZZ z 2 2 Zz z Zz zz Global Time N Z ZZVZZ Ziul 2 Global Time Z ZZ Z ZZ 2 Global Step Function Function Name Time sec Time 9 310 Set Tolerance 100th psi 20 N 10 257 Wait 40 N 11 308 Close Pressure Valve 0 N 12 257 Wait 15 N 13 307 Compare Pressures 10th psi 38 N 14 317 Save Regulator Pressure 0 N 15 310 Set Tolerance 100th psi 5 N 16 257 Wait 30 N 17 318 Compare Saved Pressure 0 N 18 309 Restore Reg Setpoint 0 N 19 68 Flush S3 15 N 20 64 Del S3 Waste 10 N 21 136 Flush Cart Solvent Block 20 N 22 259 End 0 N Bottle Change for S4 Total run time 1 15 Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 269 Set for Bottle S4 0 N 3 303 Select Regulator 6 N 4 174 Vent S4 10 N 5 176 Backflush S4 20 N 6 260 Pause for Bottle Change 0 N 7 175 Flush
96. This method can be completed in as little as 1 hour but does not allow evaluation of the chemical and amino acid background level prior to sample loading Pulsed liquid sequence a protein or peptide sample that has been applied to a precyled glass fiber disk The Pulsed liquid method includes A begin cycle to prepare the sample for sequencing A blank cycle A PTH amino acid standard cycle Sequencing cycles The Pulsed liquid method delivers a small volume of liquid TFA to the cartridge for cleavage of the ATZ amino acid Gas phase sequence a protein or peptide sample applied to a precycled glass fiber disk The Gas phase method includes A begin cycle to prepare the sample for sequencing 9 A blank cycle A PTH amino acid standard cycle Sequencing cycles The Gas phase method delivers TFA vapor to the cartridge for cleavage of the ATZ amino acid Valves Functions Cycles and Methods 3 11 Table 3 9 Standard Methods continued Method Used to PL PVDF Protein sequence a protein sample that has been applied to PVDF membrane by electroblotting or using a ProSorb device The PL PVDF Protein method includes A begin cycle to prepare the sample for sequencing A blank cycle A PTH amino acid standard cycle Sequencing cycles The PL PVDF Protein method delivers a small volume of liquid TFA to the cartridge for cleavage of the ATZ amino acid and includes a delivery of
97. Word corresponding to the greatest hazard is used Message panel which explains the hazard and any user action required Safety Alert symbol which indicates a potential personal safety hazard See the Procise Procise cLC Site Preparation and Safety Guide for an explanation of all Safety Alert symbols provided in multiple languages A waste profile was provided with this instrument and is contained in the Procise and Procise cLC Site Preparation and Safety Guide Waste profiles list the percentage compositions of the reagents within the waste stream at installation and the waste stream during a typical user application These profiles assist users in planning for instrument waste handling and disposal which must be in accordance with local state provincial or national regulations Read the waste profiles and all applicable MSDSs before handling or disposing of waste IMPORTANT Waste profiles are not a substitute for MSDS information Ensure that everyone involved with the operation of the instrument has Received instruction in general safety practices for laboratories Received instruction in specific safety practices for the instrument Read and understood all related MSDSs Me Ule Avoid using this instrument in a manner not specified by Applied Biosystems Although the instrument has been designed to protect the user this protection can be impaired if the instrument is used improperly Operating the computer correctly prevents st
98. ZZ ZZ z zz Global Step Function Function Name Time sec Time 27 148 Cartridge Wait 5 N 28 61 Del S3 Cart top 5 N 29 148 Cartridge Wait 5 N 30 61 Del S3 Cart top 5 N 31 148 Cartridge Wait 5 N 32 61 Del S3 Cart top 5 N 33 148 Cartridge Wait 5 N 34 131 Dry Cart top 120 N 35 146 Wash Large Loop Cart 10 N 36 140 Flush Large Loop Cart 20 N 37 11 Del R2g Cart top 30 N 38 140 Flush Large Loop Cart 5 N 39 6 Load RI Cart Ig loop 20 N 40 131 Dry Cart top 30 N 41 140 Flush Large Loop Cart 5 N 42 135 Flush Cart Reagent Block 5 N 43 11 Del R2g Cart top 170 N 44 132 Dry Cart bottom 30 N 45 140 Flush Large Loop Cart 5 N 46 6 Load R1 Cart Ig loop 20 N 47 140 Flush Large Loop Cart 5 N 48 135 Flush Cart Reagent Block 5 N 49 11 Del R2g Cart top 170 N 50 146 Wash Large Loop Cart 10 N 51 140 Flush Large Loop Cart 20 N 52 143 Wash Cart Reagent Block 15 N 53 135 Flush Cart Reagent Block 30 N 54 136 Flush Cart Solvent Block 30 N 55 132 Dry Cart bottom 60 N 56 63 Del S3 Cart sensor 20 N 57 131 Dry Cart top 30 N 58 63 Del S3 Cart sensor 20 N 59 131 Dry Cart top 30 N 60 53 Del S2 Cart sensor 20 N 61 51 Del S2 Cart top N 62 148 Cartridge Wait N 63 131 Dry Cart top 30 N 64 53 Del S2 Cart sensor 20 N 65 51 Del S2 Cart top 5 N 66 148 Cartridge Wait N 67 51 Del S2 Cart top N Cycle Method and Gradient Listings B 5 Step 68 69 70 71 72 73 74 75 76 Function 148 131
99. ZZ de E Bottle Change for Total run time 4 50 X1 Leak Step AN OA R 0 PM D PMO N M D N A o l SF FSF FS CE a A U N O DAN O GG A ON O Function 258 270 303 304 77 79 89 260 305 310 257 308 257 307 317 310 257 318 309 78 74 84 143 135 259 Bottle Change for X2 Total run time 2 05 Step 0 N O WG FWD Function 258 271 303 184 186 196 260 185 187 Function Name Begin Set for Bottle X1 Select Regulator Save Regulator Setpoint Vent X1 Backflush X1 Backflush X19 Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Restore Reg Setpoint Flush X1 Del X1 Waste Del X1g Waste Wash Cart Reagent Block Flush Cart Reagent Block End Function Name Begin Set for Bottle X2 Select Regulator Vent X2 Backflush X2 Backflush X2g Pause for Bottle Change Flush X2 Del X2 Waste Global Time sec Time 0 0 7 0 10 60 20 0 38 20 40 0 15 38 30 15 60 10 10 20 Z Zz Z Z Z ZZ LZ RR MS Z Z Z Z Z Z Z Z ZZ z Zz Global Time sec Time 0 0 7 10 20 20 0 15 20 Z Z Z Z Z Z Z Z Z Procedure Listings C 13 Step 10 11 12 13 Function 197 220 218 259 Bottle Change for Total run time 3 30 X2 Leak Step N DU FWD 11 12 13 1
100. a E 0 PM gt a 4 0 on Function 258 303 304 174 156 176 305 310 257 308 257 307 317 Function Name Compare Saved Pressure Vent S1 Set Tolerance 100th psi Compare Pressures 10th psi Set Reg Setpoint 10th psi Wait Close Pressure Valve Vent S2 Compare Pressures 10th psi Set Reg Setpoint 10th psi Wait Close Pressure Valve Vent S3 Compare Pressures 10th psi Restore Reg Setpoint Flush S1 Del S1 Waste Flush Cart Solvent Block Del S3 Waste Flush Cart Solvent Block Del S2 Waste Flush Cart Solvent Block End Function Name Begin Select Regulator Save Regulator Setpoint Vent S4 Backflush R4 Backflush S4 Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Time sec 0 60 10 0 38 30 1 75 0 38 30 1 60 0 0 10 10 10 10 10 10 30 0 Time sec 0 6 0 10 20 20 38 20 40 1 15 38 0 Global Time N Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Global Time Z Z Z Z Z Z Z Zz Z Z Z Z Z Procedure Listings C 37 Step 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Function 310 257 318 154 310 307 305 257 308 174 307 309 157 218 220 218 259 R5 X1 X2 Leak Test Total run time 7 15 C 38 Procedure Listings Step AN Asa 0 PM a 4 dA dA A on on oa un co O WAN O dd Aa OON O Function
101. aaa a 4 so af N O DAN DOA fa 0 ON O DAN O O BR ON O Function 258 223 303 305 303 305 303 305 303 305 303 305 303 305 303 305 303 305 94 135 341 135 333 135 286 19 287 39 342 288 89 334 337 338 339 Function Name End Function Name Begin Inject Position Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi Del X3 Waste Flush Cart Reagent Block X3 to X1 Flush Cart Reagent Block X3 to R1 Flush Cart Reagent Block X3 to R2 Backflush R2g X3 to R3g Backflush R3g X3 to X2 X3 to X1g Backflush X19 X3 to R3 X3 to S1 X3 to S2 X3 to S3 Time sec 0 Time sec Qn O A O 0 O NO O O 120 10 30 30 10 60 60 60 60 Global Time N Global Time N ZZ Z Zoe ZZZzzZ Zz Zz Zz Zz Zz ZZ ZZ zZzz z z Zz zz zz Step 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 Function 96 140 95 139 140 123 257 343 132 124 257 118 121 215 125 257 118 131 126 257 118 121 147 136 138 336 218 335 218 202 217 289 196 2
102. and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Isopropanol NUON CHEMICAL HAZARD Isopropyl alcohol can be harmful if inhaled ingested or absorbed through the skin It can cause CNS depression and be irritating to the eyes skin and mucous membranes Introduction and Safety 1 7 Table 1 2 Warnings on Chemicals Used in the Procise System continued Chemical Chemical Hazard Methanol Also in R2B CHEMICAL HAZARD Methanol is a flammable liquid and vapor Exposure may cause eye skin and respiratory tract irritation and central nervous system depression and blindness Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Piperidine hexahydropyridine CHEMICAL HAZARD Piperidine Hexahydropyridine can cause severe burns to the skin eyes and respiratory tract It is a potential reproductive toxicant potential carcinogen that can affect the heart liver and kidney It is flammable as a gas or as a liquid Avoid all skin contact with and inhalation of piperidine PMTC CHEMICAL HAZARD PMTC in acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves
103. ared Run Gradient Used in troubleshooting chromatography problems to isolate the HPLC Downloads gradient to HPLC equilibrates column and runs gradient No injection occurs 3 10 Valves Functions Cycles and Methods Methods About Methods A method combines a number of cartridge cycles flask cycles HPLC gradients and operating temperature settings in the order necessary to perform a specific task such as the analysis of a protein sample There are sequence methods for preparing glass fiber filters prior to sample loading methods specific to the sample substrate and the type of cleavage employed and methods for testing or optimizing the sequencer performance Table 3 9 Standard Methods Method Used to Filter Precycle prepare a glass fiber disk that has been treated with Biobrene Plus solution prior to applying the protein or peptide sample The Filter Precycle method includes Two filter conditioning cycles A PTH amino acid standard cycle A sequencing cycle that can be repeated a user selectable number of times allowing evaluation of the chemical and amino acid background level This method requires at least 2 5 hours for completion Fast Precycle prepare a glass fiber disk that has been treated with Biobrene Plus solution prior to applying the protein or peptide sample The Fast Precycle method includes Two filter conditioning cycles A PTH amino acid standard cycle
104. art sensor 20 N 78 51 Del 82 Cart top 5 N Cycle Method and Gradient Listings B 3 Step 79 80 81 82 83 84 85 86 87 88 Function 148 51 131 63 131 63 131 63 131 259 Cart Begin Total run time 36 55 Gas phase Step AN OOA 0 PM 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 B 4 Cycle Method and Gradient Listings Function 258 144 136 106 111 137 131 34 31 143 135 107 144 136 138 131 63 61 148 131 53 51 148 51 148 51 Function Name Cartridge Wait Del S2 Cart top Dry Cart top Del S3 Cart sensor Dry Cart top Del S3 Cart sensor Dry Cart top Del S3 Cart sensor Dry Cart top End Function Name Begin Wash Cart Solvent Block Flush Cart Solvent Block Wash Input Block S2 Wash Input Block S3 Flush Input Block Dry Cart top Del R3g Waste Del R3g Cart top Wash Cart Reagent Block Flush Cart Reagent Block Wash Output Block S2 Wash Cart Solvent Block Flush Cart Solvent Block Flush Output Block Dry Cart top Del S3 Cart sensor Del S3 Cart top Cartridge Wait Dry Cart top Del 82 Cart sensor Del 82 Cart top Cartridge Wait Del 82 Cart top Cartridge Wait Del 82 Cart top Time sec 5 5 30 20 30 20 30 20 60 0 Time sec 0 10 40 10 10 30 60 30 450 15 40 15 15 40 40 40 20 10 Global Time N Z ZIZ Z Z RZ Global Time N Z Z ZZ Z ZZ ZZ Z Z Z Z Z Zz Z Z Z
105. art sm loop Load X3 Cart Ig loop Vent X3 Cart Flush X3 Cart Backflush X3 Cart Reserved Wash Input Block S1 Wash Output Block S1 Transfer to Flask S1 Transfer to FC S1 Reserved Wash Input Block S2 Wash Output Block S2 Transfer to Flask S2 Transfer to FC S2 Reserved Wash Input Block S3 Wash Output Block S3 Transfer to Flask S3 Transfer to FC S3 Reserved Wash Input Block X3 Wash Output Block X3 Transfer to Flask X3 Valves 5 11 59 15 2 7 17 18 19 20 34 35 36 37 40 2 11 10 17 18 19 20 34 35 36 37 16 2 1 59 59 2 11 59 15 4 7 17 18 19 20 34 35 36 37 40 4 11 10 17 18 19 20 34 35 36 37 16 4 7 17 18 19 20 34 35 36 37 40 4 1 4 7 22 4 7 21 60 60 4 11 60 15 7 11 14 16 14 10 40 14 23 17 18 19 20 34 35 36 37 38 45 14 23 17 18 19 20 34 35 36 37 39 7 11 12 16 12 10 40 12 23 17 18 19 20 34 35 36 37 38 45 12 23 17 18 19 20 34 35 36 37 39 7 11 13 16 13 10 40 13 23 17 18 19 20 34 35 36 37 38 45 13 23 17 18 19 20 34 35 36 37 39 4 7 16 4 11 10 40 4 7 17 18 19 20 34 35 36 37 38 45 Global Value o o O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Sensor Function N lt Z Z Z Z lt Z Z Z Z Z Z lt lt Z lt 2222222222222 lt Z Z Z Z lt Z Z Z Z Global Time N lt
106. art top 30 N 11 140 Flush Large Loop Cart 5 N 12 135 Flush Cart Reagent Block 5 N 13 11 Del R2g Cart top 170 N 14 132 Dry Cart bottom 30 N 15 140 Flush Large Loop Cart 5 N 16 6 Load RI Cart Ig loop 20 N 17 131 Dry Cart top 30 N 18 140 Flush Large Loop Cart 5 N 19 135 Flush Cart Reagent Block 5 N 20 11 Del R2g Cart top 170 N 21 132 Dry Cart bottom 30 N 22 140 Flush Large Loop Cart 5 N 23 6 Load RI Cart Ig loop 20 N 24 131 Dry Cart top 30 N 25 140 Flush Large Loop Cart 5 N 26 135 Flush Cart Reagent Block 5 N 27 11 Del R2g Cart top 170 N 28 146 Wash Large Loop Cart 10 N 29 111 Wash Input Block S3 10 N 30 140 Flush Large Loop Cart 15 N 31 137 Flush Input Block 20 N 32 136 Flush Cart Solvent Block 30 N 33 132 Dry Cart bottom 60 N 34 142 Set Cart Temperature 48 N 35 63 Del S3 Cart sensor 15 N 36 148 Cartridge Wait 10 N 37 61 Del S3 Cart top 10 N 38 148 Cartridge Wait 10 N 39 51 Del S2 Cart top 5 N Cycle Method and Gradient Listings B 13 Step 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 B 14 Cycle Method and Gradient Listings Function 148 51 148 131 53 148 51 148 51 148 61 148 131 137 140 26 30 136 140 138 144 143 146 107 137 136 111 64 135 137 140 136 138 257 131 142 127 141 63 148 121 Function Name Cartridge Wait Del S2
107. as and X3 to the cartridge input block and to waste Cartridge solvent block Controls delivery of one reagent R3 solvents S2 S3 and S1 and argon to the cartridge reagent block cartridge input block cartridge output block and to waste Cartridge input block Controls transfer and metering of reagents solvents and argon from the cartridge reagent block and cartridge solvent block into or out of the active cartridge and to waste Cartridge output block Controls transfer of reagents solvents and argon from the cartridge reagent block and cartridge solvent block into or out of the active cartridge and to waste Flask reagent block Controls delivery and metering small loop of reagents R4 R5 X2 liquid and X3 solvent S4 and argon to the flask input block Flask input block Controls delivery X2 gas transfer and metering large loop of reagents solvents and argon from the flask reagent block to the conversion flask and to waste Flask output block Controls delivery of argon to the conversion flask for bubbling and for flushing the sample loop and transfer of the contents of the conversion flask to the sample loop and to waste The design of the valve blocks minimizes any holdup volume following chemical delivery Delivery lines feed into each valve block and connect to the common pathway manifold inside the block through a manifold inlet line and a solenoid controlled valve
108. ches it This S4B delivery minimizes exposure of the labile ATZ amino acids to the hot walls of the flask After the extractions are completed an R2B delivery to waste and a short argon dry precede an additional S2B delivery through the cartridge to the waste bottle These deliveries are essential to rinse remaining R3 from the cartridge and facilitate neutralization of the sample prior to the next sequencing cycle RI Q T C I AI A c SFaCOOH NT Ss N ont e O Heat ATZ AA PTH AA S The transferred extraction solution S3 and or S2B ATZ amino acid along with most of the initial delivery of S4B is evaporated from the conversion flask An aliguot of R4A 25 TFA in water agueous TFA is then delivered to the flask to convert the ATZ AAs to the more stable PTH AAs The presence of dithiothreitol in the R4A minimizes oxidation of the ATZ amino acids In standard sequencer cycles conversion occurs for approximately nine minutes at 64 C These conditions are sufficient to give virtually complete conversion of all amino acids except glycine about 15 of derivatized glycine remains in the intermediate PTC glycine form R5 acetonitrile is used to introduce an aliquot of the PTH standard mix to the flask for subsequent HPLC analysis After conversion is complete R4A is evaporated from the flask leaving behind the dried residue of the PTH AAs The residue is dissolved in S4B 20 aqueous acetonitrile and transferred to th
109. cid Abbreviations and Symbols Amino Acid Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Glutamine Gin Q Glutamic Acid Glu E Glycine Gly G Histidine His H Isoleucine lle Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V HPLC Solvents About the HPLC Solutions Solvent A3 Solvent B2 1012a abrf 3 See Chemical Warnings Table on page 1 7 for warnings about A3 B2 acetonitrile or the premix buffer concentrate solutions The composition of Solvent A3 is 3 5 tetrahydrofuran water Together with the Premix buffer concentrate Solvent A3 provides optimal separation of the PTH amino acids Solvent B2 contains 12 isopropanol in acetonitrile to resolve PTH Trp from the chemical artifact diphenylurea DPU 2 Standard 1 T dptu dmptu 6 0 9 0 12 0 15 0 18 0 Figure 7 1 5 pmoles of PTH amino acid standard was analyzed using solvent B2 and solvent A3 containing Premix Buffer Concentrate Notice that the DPU is favorably positioned between Trp and Phe PTH Separation 7 3 Premix Buffer Concentrate Adding Premix Buffer 7 4 PTH Separation The
110. ct the specific cycle to be copied 4 Use the File menu from the upper menu bar to select Save Cycle Procedures As 5 Type the new cycle name and click the OK button Editing the Copied To edit the copied cycle Flask Cycle Saving an Edited Flask Cycle Step Action 1 To delete a row highlight the row to be deleted and click the Delete Row button 2 To insert a row select the function to be inserted from the function list on the left side of the screen The function can be selected by either using the scroll down button or typing the function number at the top corner of the function list a Highlight the row immediately before the intended insertion point b To enter the function run time click on the global box to turn the global time off Type the function time in seconds in the value box To save the edited cycle Step Action 1 Pull down the File menu from the upper menu bar 2 Select Save Cycle Procedures IMPORTANT The Ready to Receive step in a Flask cycle synchronizes with the Ready to Transfer Step in a Cartridge Cycle The flask cycle must have a Ready to Receive step to receive sample from the cartridge The Prepare Pump step initiates the pump to start running and equilibrates the column at the initial condition of the gradient to be run Allow at least 8 minutes between the Prepare Pump and Load Injector steps The maximum number of steps allowed per cycle is 100
111. ction 258 267 303 304 57 59 260 305 310 Function Name Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Restore Reg Setpoint Flush S1 Del S1 Waste Flush Cart Solvent Block Wash Cart Solvent Block Flush Cart Solvent Block End Function Name Begin Set for Bottle S2 Select Regulator Vent S2 Backflush S2 Pause for Bottle Change Flush S2 Del S2 Waste Flush Cart Solvent Block End Function Name Begin Set for Bottle S2 Select Regulator Save Regulator Setpoint Vent S2 Backflush S2 Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Global Time sec Time 38 30 15 10 10 20 N Z Z Z Z Z Z Z Z Z Z Z Global Time sec Time 0 0 4 10 20 0 15 10 20 0 Z Z Z Z Z Z Z Z Z Z Global Time sec Time 0 0 4 0 10 20 0 38 20 N ZZZ Procedure Listings C 9 Step 10 11 12 13 14 15 16 17 18 19 20 21 22 Function 257 308 257 307 317 310 257 318 309 58 54 136 259 Bottle Change for S3 Total run time 1 15 Step O ON OOO ADN Function 258 268 303 67 69 260 68 64 136 259 Bottle Change for Total run time 2 40 S3 Leak C 10 Procedure Listings Step O MN o RAO PM Function 258 268 303 304 67 69 260 305 Function Name Wait Close Pressure Valve Wait Compare Pressures 10th psi Save
112. ction Name Time sec Time 21 135 Flush Cart Reagent Block 10 N 22 143 Wash Cart Reagent Block 10 N 23 135 Flush Cart Reagent Block 20 N 24 259 End 0 N Bottle Change for R2 Total run time 1 00 Global Step Function 4 Function Name Time sec Time 1 258 Begin 0 N 2 262 Set for Bottle R2 0 N 3 303 Select Regulator 2 N 4 17 Vent R2g 10 N 5 19 Backflush R2g 10 N 6 260 Pause for Bottle Change 0 N 7 18 Flush R2g 15 N 8 143 Wash Cart Reagent Block 5 N 9 135 Flush Cart Reagent Block 20 N 10 259 End 0 N Procedure Listings C 3 Bottle Change for Total run time 2 00 R2 Leak Step O MN OA ADN ND N N a do CEC N O O JO oa ON Function 258 262 303 304 17 19 260 305 310 257 308 257 307 317 310 257 318 309 18 143 135 259 Bottle Change for R3 Total run time 3 35 C 4 Procedure Listings Step AN OA E 0 PM gt a N 2 o Function 258 263 303 304 27 29 39 260 305 28 24 34 Function Name Begin Set for Bottle R2 Select Regulator Save Regulator Setpoint Vent R2g Backflush R2g Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Restore Reg Setpoint Flush R2g Wash Cart Reagent Block Flush Cart Reagent Block End Function Name Begin Set for Bottle R3 Select Regulator
113. ction for 40 seconds to make sure that the sensor light path is clear of any liquid 4 a Activate the Deliver to Cartridge sensor function for the bottle or solvent of choice For example select Function 75 to deliver reagent or solvent from the X1 bottle position to the cartridge outlet sensor b Watch for the appearance of a check mark next to the reaction flow sensor field at the top of the screen c Note the elapsed time and add 5 10 seconds Enter this load time in the cycle for this function d Select All Off before leaving the Manual Control view 5 From the Function view enter the load time in the global time field for that function Once the time for loading the large loop has been determined the same value can be used for loading the small loop Rules to Apply to The following rules apply to using cartridge load functions in a custom cycle Using Cartridge The cartridge must be flushed for at least 40 seconds before the first delivery Load Functions E dai The cartridge must be flushed for at least 40 seconds between deliveries 6 14 Chemical Optimization Chemistry Optimization Overview Cleavage Efficiency Amino Acid Background This section helps explain the various processes used during Edman chemistry and what is necessary for the creation or optimization of chemistry cycles See Chemical Warnings Table on page 1 7 for warnings about reagents or solutions mentioned in the following pa
114. cycles Method To start the run Step Action 1 Click Start Run The Init Sensor procedure begins You can click Jump to advance to the last step of this procedure 2 Once the cartridge and flask have reached the proper temperatures the Flask Optimization method begins 3 At the first pause check the flask for liquid The flask should not be completely dry If the flask is not dry proceed to step 4 If Then the flask is dry check the pressure settings on the protein sequencer which should be set to the default values the pressure settings are not reset them to the default settings and start correct the procedure over Click Resume 5 Click Hold as soon as the Pre Conversion step begins When the flask contents visibly stop bubbling when there are between 5 and 10 uL of liquid remaining in the flask note both the Time and Remaining values shown Be sure to mark these as Pre Conversion Dry values To continue the run Step Action 1 Click Next Step twice 2 At the next pause click Resume 3 Click Hold as soon as the Dry Flask step begins 4 When the flask is visibly dry note both the Time and Remaining values Be sure to mark these as Post Conversion Dry values To calculate the optimized pre and post conversion dry step times Step Action 1 Calculate the optimum time for the Pre Conversion Dry step by
115. d Cycle Del S4 Flask Dry Flask Empty Flask Del R4 Flask Dry Flask Empty Flask Flush Large Loop Flask Wait Load R5 Flask Ig loop Dry Flask Pre Conversion Dry Flush Large Loop Flask Load R4 Flask Ig loop Dry Flask Flush Large Loop Flask Load S4 Flask Ig loop Flush Large Loop Flask Wait Post Conversion Dry Load Position Prepare Pump Time sec 10 40 1 15 10 5 10 10 10 5 30 10 0 Time sec 0 0 15 10 20 15 10 20 10 150 20 60 0 10 20 10 10 15 10 540 Global Time N Zz 2 2 LL Zz Z Z Zz ZZ Global Time Z Z Zox Zo L ZZZKxXZzzZzzizizzz zz z Global Step Function Function Name Time sec Time 24 213 Dry Flask 150 N 25 257 Wait 300 N 26 173 Load S4 Flask Ig loop 15 N 27 213 Dry Flask 10 N 28 218 Flush Large Loop Flask 10 N 29 173 Load S4 Flask lg loop 15 N 30 213 Dry Flask 10 N 31 218 Flush Large Loop Flask 10 N 32 221 Flush Injector 30 N 33 257 Wait 5 N 34 221 Flush Injector 30 N 35 213 Dry Flask 5 N 36 238 Concentrate Sample 0 Y 37 257 Wait 10 N 38 225 Load Injector 40 N 39 249 Inject Pos Collect Data 1 N 40 171 Del S4 Flask 15 N 41 213 Dry Flask 10 N 42 212 Bubble Flask 5 N 43 215 Empty Flask 10 N 44 171 Del S4 Flask 10 N 45 213 Dry Flask 10 N 46 212 Bubble Flask 5 N 47 222 Flush Flask Injector 30 N 48 221 Flush Injector 10 N 49 259 End 0 N Flask Normal Total run time 31 38 Global Step Function Function Name Time sec Time 1
116. d the proper temperatures the Flask Optimization method begins 3 At the first pause Click Resume 4 At the next pause check the flask for liquid The flask should still contain 5 10 uL of liquid approximately 1 3 of the conical section of the flask At the next pause click Resume and continue watching the liquid dry in the flask Once the flask is visibly dry note the amount of time that elapses from this point to the end of the step The elapsed time should be approximately 200 sec 7 Click Stop Run to end the cycle IMPORTANT If the verification was not successful perform the optimization procedure again Optimizing Sensor Functions About the Sensors Sensor functions are specialized valve controlling functions These functions are controlled by the target sensor rather than by a specific time setting When a function is activated the sensor begins looking for fluid When fluid is detected by the sensor the reagent or solvent delivery valve is turned off The time for the function continues to count down to zero and the next step begins In order for a sensor function to perform correctly enough time must be allotted in the step for fluid to reach the sensor If fluid reaches the sensor within the allotted step time there is no report to the event log If fluid does not reach the sensor within the allotted time an error will be reported in the Event Log and the protein sequencer may be paused Each optical
117. dy to Receive step 4 8 Creating Functions Cycles and Methods Tests and Procedures Overview About This Chapter In addition to sequencing cycles and methods the Procise system provides a number of test cleanup and diagnostic procedures An explanation of the setup and execution of each test as well as the method for creating new tests and procedures is in this chapter A complete listing of the tests and procedures can be found in Appendix C In This Chapter This chapter contains the following topics Topic See Page Running Tests 5 2 Flow Test Procedures 5 3 Startup Procedures 5 4 Idle Procedures 5 5 Init Sensor Procedures 5 5 Leak Procedures 5 6 Shutdown Procedures 5 7 Cleanup Procedures 5 8 Electrical Test Procedure 5 8 Bottle Change Procedures 5 9 Creating Tests and Procedures 5 11 Tests and Procedures 5 1 Running Tests Using Test All available tests and procedures can be run from the Tests view except bottle change Procedures procedures Run bottle change procedures from the Bottle Change view IMPORTANT Tests and Procedures can not be run while the sequencer is active To use a test procedure Step Action 1 Select the Tests View from the View pop up menu 2 Select a type of test by clicking the appropriate radio button 3 If the Then Don t pause at error box is the designated test s will be completed checked regardle
118. e This height depends upon the physical proportions of the user Adjust vision factors to optimize comfort and efficiency by Adjusting screen variables such as brightness contrast and color to suit personal preferences and ambient lighting Positioning the screen to minimize reflections from ambient light sources Positioning the screen at a distance that takes into account user variables such as nearsightedness farsightedness astigmatism and the effects of corrective lenses When considering the user s distance from the screen the following are useful guidelines The distance from the user s eyes to the viewing screen should be approximately the same as the distance from the user s eyes to the keyboard For most people the reading distance that is the most comfortable is approximately 20 inches The workstation surface should have a minimum depth of 36 inches to accommodate distance adjustment Adjust the screen angle to minimize reflection and glare and avoid highly reflective surfaces for the workstation Use a well designed copy holder adjustable horizontally and vertically that allows referenced hard copy material to be placed at the same viewing distance as the screen and keyboard Keep wires and cables out of the way of users and passersby Choose a workstation that has a surface large enough for other tasks and that provides sufficient legroom for adequate movement Introductio
119. e sequence may be the primary reason for a sample to stop producing useful sequence data Repetitive exposure of the sample to strong acid during the cleavage reaction can cause cleavage between amino acids elsewhere in the peptide chain Each time non specific cleavage of the peptide chain occurs a new N terminus is generated which can react with PITC This will cause an increase in the amino acid background the presence of other PTH amino acids in the chromatogram which do not reflect the true N terminal sequence At the start of a sequencing run the amino acid background from non specific cleavage is low This background increases with each sequencing cycle Fortunately non specific cleavage is sequence specific so only peptide bonds between amino acids will be cleaved This keeps the amino acid background rate from cycle to cycle quite low However for proteins with labile amino acid sequences and very large proteins amino acid background will increase much more rapidly In practical terms while 50 picomoles of a 100 200 amino acid protein may provide 40 50 cycles of interpretable sequence the same amount of a 2000 amino acid protein will probably provide only 10 15 cycles of sequence Chemical Optimization 6 15 Coupling About the Coupling Process PITC Delivery Coupling Base Delivery 6 16 Chemical Optimization Coupling is the process of reacting the free amino terminus of a protein or peptide sample with P
120. e HPLC system In standard sequencer cycles two metered volumes of S4B are delivered to the flask mixed and allowed to incubate to ensure complete dissolution The solution is then transferred to the HPLC system Subsequent S4B deliveries are made to pick up any residual PTH amino acid and to rinse the lines between the conversion flask and the injector In preparation for the next conversion S4B is then delivered to fill the flask mixed by argon bubbling and emptied to waste Sequence Analysis Chemistry 2 5 Separation and The HPLC pump forces the sample mixture and mobile phase through the column Analysis of Under pressure The various components of the sample having different relative PTH AAs affinities for the stationary phase inside the column will exit the column at different times corresponding to different solvent strengths The pump flow rate the solvent composition and the oven temperature determine the speed and order of elution of the PTH AAs in the sample The mobile phase amino acid mixture travels from the end of the column into a detector flowcell The detector measures the UV light absorbance of the column effluent and the analog signal is output to external recording devices 2 6 Sequence Analysis Chemistry Valves Functions Cycles and Methods Overview About This Chapter This chapter defines the components that make up a sequencing run In This Chapter This chapter contains the following topics
121. e allowed per method A cartridge cycle containing a Transfer to Flask step must be matched with a flask cycle containing a Ready to Receive step Creating Functions Cycles and Methods 4 7 Creating New To create a new method Methods Step Action 1 Use the View menu to select Sequence Methods View 2 Use the method screen menu to select the User Defined method 3 Create the new method a Highlight the default method row Select the new cartridge cycle flask cycle and gradient from each pop up menu To delete a row highlight the row to be deleted and click the Delete button c To add a row highlight the row after which the new row will be inserted and click the Insert Row button Move the cursor to the cycle field and enter the cycle number to be added as an exception Select the proper cartridge cycle flask cycle and gradient for the cycle from each pop up menu Saving the New To save the new method Method Step Action 1 Pull down the File menu from the upper menu bar 2 Select Save Methods As 3 Type the new name and click the OK button IMPORTANT A method must contain a valid default cycle which is the cycle that is run when there is no exception cycle The default cycle is not necessarily a canned cycle A cartridge cycle containing a Ready to Transfer step must be matched with a flask cycle containing a Rea
122. ed in Applied Biosystems printed product literature or this Warranty Statement Any such affirmation representation or warranty made by any agent employee or representative of Applied Biosystems will not be binding on Applied Biosystems Warranty E 1 E 2 Warranty Applied Biosystems shall not be liable for any incidental special or consequential loss damage or expense directly or indirectly arising from the purchase or use of the Instrument Applied Biosystems makes no warranty whatsoever with regard to products or parts furnished by third parties This Warranty is limited to the original location of installation and electrical power connection and is not transferable THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS Index Numerics 1 chlorobutane see also S3 2 4 20 acetonitrile see also S4B 2 5 25 aqueous TFA see also R4A 2 5 A A3 solvent 7 3 acetone changing baseline 7 7 acetonitrile see also R5 2 5 amino acid background 6 15 table of abbreviations 7 2 arginine 7 8 argon delivery valves 3 5 flushing system 5 8 storing with 2 3 aspartic acid 7 9 modified 6 16 aspartic acid separating 7 9 ATZ anilinothiazolinone extraction describing 6 19 transfer 2 4 B
123. el X2 Flask Load X2 Flask sm loop Load X2 Flask Ig loop Vent X2 Flush X2 Backflush X2 Del X2 Waste Reserved Reserved Reserved Del X2g Flask Not Available Valves 51 51 28 51 24 28 31 29 32 45 29 30 29 31 49 49 29 49 24 29 31 25 32 45 25 30 25 31 50 50 25 50 24 25 31 27 32 45 27 30 27 31 57 57 27 57 24 27 31 33 32 45 Global Value o o O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Sensor Function N lt Z Z Z Z Z Z ZZ lt lt Z Z Z Z Z Z Z Z lt lt Z Z Z Z Z Z Z 2 Z Z Z Z Z Z Z Z lt Global Time Y lt lt Z Z Z Z lt lt lt lt Z Z Z Z lt lt x x lt lt Z Z Z Z lt lt lt lt Z lt Z Z Z Z lt lt lt lt Number 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 Reagent X2g X2g X2g X2g X2g X2g X2g X2g X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 Name Not Available Vent X2g Flush X2g Backflush X2g Del X2g Waste Reserved Reserved Reserved Del X3 Flask Load X3 Flask sm loop Load X3 Flask Ig loop Vent X3 Flask Flush X3 Flask Backflush X3 Flask Del X3 Waste Flask Reserved Reserved Reserved Bubble Flask h press Bubble Flask Dry Flask Dry Flask h press Empty Flask Empty Flask
124. en Valves 49 57 59 Waste Test Pause Use Valves of Function Select Regulator Save Regulator Setpoint Set Reg Setpoint 10th psi Wait With Valves On Valves 11 1511 15 24 32 41 42 47 3 4 58 4 9 11 53 2 4 59 26 33 57 7 11 15 16 24 30 47 43 44 15 23 24 24 32 24 32 45 44 24 47 49 57 59 43 44 Global Value 0 o O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Sensor Function N lt lt lt lt lt lt lt lt z z Z ZZ ZZZzZzZZ Zz Z Z Zz ZZz Z Zz Z ZZ Z Z Z xX AK Function Listing Global Time N Z 2 z ZZ Z Z Z ZZ Z Z zz Z Z z Z ZZ Z Z Z Z Z Zz Zz Z Z Z Zz Z Z Z Z A 9 Number Reagent 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 Name Valves Compare Pressures 10th psi Close Pressure Valve Restore Reg Setpoint Set Tolerance 100th psi Test Valves Test Heaters Test Pressure Board Test 12 Bit A D Test 24 Bit A D Test Rheodyne Save Regulator Pressure Compare Saved Pressure Compare HP Inlet 10th psi Select Heater Save Heater Setpoint Restore Heater Setpoint Inc Heater Setpoint C Dec Heater Setpoint C Set Heater Tolerance 100th C Compare Temperatures Reset Vacuum On Count Log Vacuum On Count Set Flow Meter Tolerance SCCM Compare
125. ensor Cartridge Wait Del S2 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Del S3 Cart top Cartridge Wait Dry Cart top Flush Input Block Flush Large Loop Cart Load R3 Cart Ig loop Transfer R3 Cart gas Flush Cart Solvent Block Flush Large Loop Cart Flush Output Block Wash Cart Solvent Block Wash Cart Reagent Block Wash Large Loop Cart Wash Output Block S2 Flush Input Block Flush Cart Solvent Block Time sec 5 5 170 10 10 15 20 30 60 48 15 10 10 10 5 Global Time N Z Zz Z Ziz Z Zz ZZZ ZZ ZZ Zz ZZ Zz Z ZZ LL Ze Zz ZZ Z Z Z Z z Z Global Step Function 4 Function Name Time sec Time 62 111 Wash Input Block S3 5 N 63 64 Del S3 Waste 5 N 64 135 Flush Cart Reagent Block 40 N 65 137 Flush Input Block 30 N 66 140 Flush Large Loop Cart 30 N 67 136 Flush Cart Solvent Block 60 N 68 138 Flush Output Block 30 N 69 257 Wait 0 Y 70 131 Dry Cart top 40 N 71 142 Set Cart Temperature 48 N 72 127 Ready Transfer to Flask 0 N 73 141 Flush Transfer Line 5 N 74 63 Del S3 Cart sensor 15 N 75 148 Cartridge Wait 10 N 76 121 Transfer to Flask gas 45 N 77 141 Flush Transfer Line 5 N 78 53 Del S2 Cart sensor 15 N 79 148 Cartridge Wait 10 N 80 121 Transfer to Flask gas 45 N 81 141 Flush Transfer Line 5 N 82 63 Del S3 Cart sensor 15 N 83 148 Cartridge Wait 10 N 84 121 Transfer to Flask gas 45 N 85 128 Transfer Complete 0 N 86 131 Dry Cart top 60 N 87 61 Del S3 Car
126. ensures the leak tightness of each cartridge It pressurizes the cartridge and monitors the leak rate The test result will be reported in the Event Log at the end of the test The actual cartridge pressure must be within 0 1 psi of the target pressure in order to pass the cartridge leak test The flask leak test checks for leak tightness and venting capability of the flask assembly The test result will be reported in the Event Log at the end of the test The actual flask pressure must be within 0 05 psi of the target pressure in order to pass the flask leak test The valve block leak tests check the leak tightness and venting capability of each valve block The actual pressure held in the valve block must be within 0 05 psi of the target pressure in order to pass the valve block leak test Shutdown Procedures Shutdown Procedures Post Run Valve Block Wash X3 Short Term Shutdown Procedure Shutdown procedures can be programmed from the Start Run view for the automatic run The shutdown procedure is executed after completion of the last cartridge scheduled to be run Two shutdown procedures are permanently stored in the Procise sequencer software Post Run Valve Block Wash X3 Short Term Shutdown Procedure This procedure is used to wash the system flowpaths from the X3 bottle position Methanol should be loaded in the X3 position before starting this procedure See Chemical Warnings Table on page 1 7 for warnings about
127. epetitive cleavage reactions Cart Begin Prepares sample for pulsed liquid sequencing by delivering an aliquot of TFA to denature the sample followed by coupling with PITC Cart Begin Gas phase prepares sample for sequencing by delivering TFA vapor to Gas phase denature the sample followed by coupling with PITC Cart Edman chemistry cycle for sequencing samples on polybrene coated Pulsed liquid glass fiber disks Delivers an aliquot of liquid TFA for cleavage Three ATZ extractions two with butyl chloride one with ethyl acetate Cart Edman chemistry cycle for sequencing samples on polybrene coated Gas phase glass fiber disks Delivers TFA vapor for cleavage Three ATZ extractions two with butyl chloride one with ethyl acetate Cart PL PVDF Protein Edman chemistry cycle for sequencing protein samples on PVDF membrane Delivers an aliquot of liquid TFA for cleavage Includes pre wetting of the membrane with methanol water X1 bottle prior to coupling and an additional extraction of the membrane after cleavage Three ATZ extractions two with butyl chloride one with ethyl acetate Cart PL PVDF Edman chemistry cycle for sequencing peptide samples on PVDF Peptide membrane Delivers an aliquot of liquid TFA for cleavage Three ATZ extractions two with butyl chloride one with ethyl acetate Cart GP PVDF Edman chemistry cycle for sequencing protein samples on PVDF Protein membrane Delivers TFA vap
128. es 10 Method Injector Optimization To run the optimization procedure Step Action 1 Click Start Run The Init Sensor procedure will start running You can click Jump to advance to the last step of this procedure 2 If the flask temperature is 64 C when the run pauses at the Begin step of the flask cycle click Next Step to start the Injector Optimization method 3 Click Pause Later and configure the run to pause on Cartridge A at the end of the first cycle 4 At the end of the first cycle look for the injection slug in the plumbing line Ideally the end of the slug should be between the mark you made on the tubing and the valve block It should not be in the pickup line connected to port 41 5 If the end of the injection slug is not in the correct location modify the Concentrate Sample time global value in the Functions dialog box Increase or decrease the value as appropriate in 5 sec increments only IMPORTANT Do not increase the Concentrate Sample step time by more than 5 sec at a time Otherwise an air injection might occur An air injection will damage the column 6 Click Resume Once you have determined the correct value for the Concentrate Sample step run at least one more cycle to confirm the optimization Chemical Optimization 6 3 Optimizing Flask Dry Times Overview Set up a protein sequencer run to optimize the global time values for the Pre and
129. est Procedure C 25 Flow Procedure List C 26 Idle Procedure C 29 Init Sensor Procedure C 30 Leak Procedure List C 32 Shutdown Procedure List C 45 Startup Procedure C 47 Procedure Listings C 1 Bottle Change Procedure List Bottle Change for R1 Total run time 1 35 C 2 Step MN O MAO PM gt A N o Function 258 261 303 Bottle Change for Total run time 2 45 R1 Leak Procedure Listings Step AN Oa R ON 11 12 13 14 15 16 17 18 19 20 Function 258 261 303 304 7 9 260 305 310 257 308 257 307 317 310 257 318 309 Function Name Begin Set for Bottle R1 Select Regulator Vent R1 Backflush R1 Pause for Bottle Change Flush R1 Del R1 Waste Flush Cart Reagent Block Wash Cart Reagent Block Flush Cart Reagent Block End Function Name Begin Set for Bottle R1 Select Regulator Save Regulator Setpoint Vent R1 Backflush R1 Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Compare Pressures 10th psi Save Regulator Pressure Set Tolerance 100th psi Wait Compare Saved Pressure Restore Reg Setpoint Flush R1 Del R1 Waste Time sec 0 0 1 10 20 0 15 10 10 10 20 0 Time sec 0 0 1 0 10 20 0 38 20 25 0 15 38 0 5 30 0 0 15 10 Global Time Z Z Zi ZZZ zz zz Global Time Z Z Z Z ZZ ZZZ Z Z Z ZKZ ZA Z Z Global Step Function Fun
130. etting Help Technical Support Contacting Technical Support To Contact Technical Support by E Mail Hours for Telephone Technical Support You can contact Applied Biosystems for technical support by telephone or fax by e mail or through the Internet You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems Web site please see the section To Obtain Documents on Demand following the telephone information below Contact technical support by e mail for help in the following product areas Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystems com Sequence Detection Systems and PCR pcrlab appliedbiosystems com Protein Sequencing Peptide and DNA Synthesis corelab appliedbiosystems com Biochromatography PerSeptive DNA PNA and Peptide Synthesis systems CytoFluor FMAT Voyager and Mariner Mass Spectrometers tsupport appliedbiosystems com LC MS Applied Biosystems MDS Sciex apisupport sciex com or api3 support sciex com Chemiluminescence Tropix tropix appliedbiosystems com In the United States and Canada technical support is available at the following times Product Hours Chemiluminescence 8 30 a m to 5 30 p m Eastern Time Framingham
131. ient and Programming Fast Gradient The fast gradient is programmed in the Procise software It provides reduced chromatographic turnaround time through increased mobile phase flow rate The gradient programmed in Procise software is the linear gradient which provides flattened baseline throughout the chromatography However any fast gradients published previously in User Bulletins or User s Manuals should provide satisfactory separation with this buffer system Table 7 2 Fast Gradient Conditions Topic Description Target Pressure 1000 psi Target Time 1 0 minutes Pressure Limits 0 4000 psi Flow rate Step Time min B HL min Events 0 6 325 12 2 0 3 6 325 1 3 0 4 16 325 1 4 18 45 325 1 5 18 5 90 325 1 6 21 5 90 325 1 7 22 50 325 1 Separating Adding a gradient step at 0 4 minutes and increasing the solvent B concentration PTH derivatives 3 5 over initial concentration can provide improvement in the separation of the Gln Thr and Gly PTH derivatives of Gin Thr and Gly This modification to the gradient allows the initial B to be lower in order to improve the retention of PTH Asp on the column without compromising the separation of DMPTU from PTH Glu PTH Separation 7 5 Changing an Active The gradients programmed in Procise software can be edited without having to first Gradient Save them under another name The active gradient can be changed during a run and
132. installed Table 5 1 Flow Test Procedure Flow Test Usage Sensor amp Delivery Test Check sensor activities This procedure tests the operation of 11 fluid optical sensors It delivers the chemical from the bottle through specific sensors and checks whether or not fluid was sensed within a function time If fluid was not sensed either the sensor is faulty or the delivery was incomplete Any failures are reported in the event log Prior to starting this test it might be necessary to run the Init Sensor procedure to set up the sensors for operation if it has not been run automatically as a part of the method Note The test should be run while the designated sequencing chemicals are loaded on the instrument In addition X1 must contain 50 50 methanol water X2 must contain methanol X3 must contain methanol See Chemical Warnings Table on page 1 7 for warnings about methanol solutions Once the procedure is complete select the Event Log view from the View pop up menu to determine whether any delivery errors have occurred Tests and Procedures 5 3 Startup Procedures About the Startup A startup procedure is permanently stored in the Procise software The startup Procedure procedure flushes each reagent solvent bottle with argon and refreshes the reagent in the delivery line The flask is washed with S4 No solvent or reagent is delivered through the cartridges See Chemical Warnings Table o
133. ion Drying PTH amino Acid Solubilization Sample Transfer and Injection Sample Volume 6 20 Chemical Optimization conversion instead of the large load This would reduce the amount of drying necessary after conversion After conversion the sample must be completely dried to remove all the TFA which will interfere in the chromatography of the early eluting PTH amino acids In the standard flask cycles the flask will appear dry 90 120 seconds before the end of the drying step which follows the Post conversion Dry step This will not adversely affect the recovery of the PTH amino acids To simplify optimization of this drying step function 237 Post conversion Dry has a global time setting Changing the global time setting in the Function view will adjust the length of this step in any cycles in which it is used See Setting Global Time on page 4 3 for instructions on modifying a global time setting The dried PTH amino acid in the flask is dissolved in 20 acetonitrile S4 for subsequent transfer to the injector loop Two large loop loads of S4 are used to dissolve the sample in the standard flask cycles Bubbling of the flask contents assists the dissolution of the sample Once the sample has been reconstituted in the flask it must be transferred to the HPLC injector loop Transfer is accomplished by pressurizing the flask with argon and driving the sample out through the pick up line and into the injector loop When the sa
134. ions can be found in Creating User Defined Functions on page 4 2 3 8 Valves Functions Cycles and Methods Cycles About Cycles To perform complex chemical tasks groups of functions are organized together into Standard Cartridge Cycles cycles defined here in Table 3 6 Once incorporated into a cycle individual functions are referred to as steps of that cycle and will be activated for a specified period of time during that cycle Table 3 6 Defining the Cycle Types Cycle Type Definition Cartridge cycles blocks are referred to as cartridge cycles Chemical processes occurring in the glass reaction cartridge Flask cycle referred to as flask cycles Processes that take place in the conversion flask are Standard cycles are referred to as standard cycles The permanent cycles provided with the protein sequencer Note These cycles can not be deleted or modified but can be used as templates for creating new cycles after the cycle has been stored under a new name The specifics of creating new cycles are described in Creating Cycles on page 4 4 There are nine standard cartridge chemistry cycles A complete cartridge cycle listing can be found in Appendix B Cycle Method and Gradient Listings Table 3 7 Standard Cartridge Cycles Cycle Description Cart Precycle Prepares a polybrene treated glass fiber disk for sequencing by running abbreviated coupling and r
135. is flushed with argon gas a BB OJN The chemical is delivered to the waste bottle and washes the associated valve blocks and Teflon lines Two procedures are available for each bottle position A bottle change only A leak test on the bottle position as well as a bottle change Note The bottle change procedure can only be used when the Procise system is idle or paused To start the bottle change procedure Step Action 1 Select the Bottle Change view from the View pop up menu 2 Highlight the desired procedure from the bottle change procedure pop up menu 3 Click the OK button Table of Bottle Table 5 2 Bottle Change and Cycle Time Changes Procedure Cycle Time min Bottle Change for R1 1 35 Bottle Change for R1 leak 2 45 Bottle Change for R2 1 00 Bottle Change for R2 leak 2 00 Bottle Change for R3 3 35 Bottle Change for R3 leak 4 50 Bottle Change for R4 1 35 Bottle Change for R4 leak 3 00 Bottle Change for R5 1 25 Bottle Change for R5 leak 2 50 Bottle Change for S1 1 30 Bottle Change for S1 leak 2 55 Bottle Change for S2 1 15 Bottle Change for S2 leak 2 40 Bottle Change for S3 1 15 Bottle Change for S3 leak 2 40 Tests and Procedures 5 9 Table 5 2 Bottle Change and Cycle Time continued Procedure Cycle Time min Bottle Change for S4 1 15 Bottle Change for S
136. k 5 N 3 11 Del R2g Cart top 30 N 4 140 Flush Large Loop Cart 10 N 5 6 Load R1 Cart Ig loop 20 N 6 131 Dry Cart top 30 N 7 140 Flush Large Loop Cart 5 N 8 135 Flush Cart Reagent Block 5 N 9 11 Del R2g Cart top 170 N 10 132 Dry Cart bottom 30 N 11 140 Flush Large Loop Cart 5 N 12 6 Load R1 Cart Ig loop 20 N 13 131 Dry Cart top 30 N 14 140 Flush Large Loop Cart 5 N 15 135 Flush Cart Reagent Block 5 N 16 11 Del R2g Cart top 170 N 17 132 Dry Cart bottom 30 N 18 140 Flush Large Loop Cart 5 N 19 6 Load RI Cart Ig loop 20 N 20 131 Dry Cart top 30 N 21 140 Flush Large Loop Cart 5 N 22 135 Flush Cart Reagent Block 5 N 23 11 Del R2g Cart top 170 N 24 146 Wash Large Loop Cart 10 N 25 111 Wash Input Block S3 10 N 26 140 Flush Large Loop Cart 15 N 27 137 Flush Input Block 20 N 28 136 Flush Cart Solvent Block 30 N 29 132 Dry Cart bottom 60 N 30 142 Set Cart Temperature 48 N 31 63 Del S3 Cart sensor 15 N 32 148 Cartridge Wait 10 N 33 61 Del S3 Cart top 10 N 34 148 Cartridge Wait 10 N 35 51 Del S2 Cart top 5 N 36 148 Cartridge Wait 5 N 37 51 Del S2 Cart top 5 N 38 148 Cartridge Wait 5 N 39 131 Dry Cart top 30 N Cycle Method and Gradient Listings B 11 Step 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 B 12 Cycle Method and Gradient Listings Function 53 148 51 148 51 148 61
137. k temperature at a fixed time during a cycle Acceptable temperature range ambient to 78 C Prepare Pump 227 Downloads gradient information from the Procise software to the pump After downloading is complete 30 60 sec the pump will start pressurize and run at the initial gradient conditions Stop Pump 231 Stops any pump activity Start Gradient 232 Used to start the gradient in cases where there will be no sample injected Inject Position 223 Switches the sample loop into the HPLC flow path Not necessary when using the Sample Loop Load sensor When the sensor detects fluid the Rheodyne valve is automatically activated Set Column Temperature 229 Used to adjust the column temperature at a fixed time during the flask cycle Acceptable temperature range ambient to 70 C Valves Functions Cycles and Methods 3 7 Table 3 5 Common Functions Function Name Number Description Begin 258 This function must be the first step of all cycles tests and procedures End 259 This function must be the last step of all cycles tests and procedures Wait 257 This function is used to keep the cycle time running for a particular step in a cycle while all the valves are closed User Functions User functions can be created and named for specialized needs or applications Fifty potential functions are available An explanation of creating user funct
138. large Sensor The protein sequencer will pause at end of this cycle Dry 500 Threshold 750 Average wet xx dry wet dry wet dry wet xx XX XX XX XX XX XX XX XX XX XX XX XX XX XX XX XX XX The information provided by the Event Log is as follows The date and time of the failure The step number and cycle number of the failure and the name of the sensor reporting the failure Whether or not the protein sequencer will be paused The next entries are Dry 500 Threshold 750 Average wet xx Item Description Dry 500 The empty tube transmission dry reading from the sensor generated during the Init Sensor procedure Threshold 750 The minimum transmission value necessary for a sensor reading to be considered wet dry reading multiplied by 1 5 Average wet xx Actual transmission reading with fluid in tube if no fluid is detected average wet 0 Error Message Detecting No Flow Error Message Detecting Bubbles The last entry table is dry wet dry wet dry wet XX XX XX XX XX XX XX XX XX XX XX XX XX XX XX XX XX xx These values represent the number of dry and wet readings taken by the sensor Each sensor requires a certain number of wet readings to discriminate the arrival of the expected reagent or solvent from a stray droplet of fluid in the line If fluid never reaches the sensor only the first dry field will have a non zer
139. leucine 7 10 M manifold design 3 3 Manual Injection method 3 13 B 33 methione separating from valine 7 10 method copying existing 4 7 creatinganew 4 8 defininga 3 2 definition and table 3 11 table of names temperature B 32 O optimizing 6 4 to 6 6 cartridge load functions 6 10 chemistry 6 1 to 6 21 chromatography 7 7 to 7 11 cleavage 6 18 conversion 6 19 deliver to cartridge sensor functions 6 14 deliver to cartridge sensor function time 6 14 drying after cleavage 6 18 drying after coupling 6 17 drying flask contents 5 3 edman chemistry overview 6 15 flask chemistry 6 19 flask dry times 6 4 flask load functions 6 12 flask opt list C 26 gas phase cleavage 6 18 injection percentage 6 21 injector 6 2 to 6 3 membrane bound samples 6 19 post conversion drying 6 20 post coupling wash 6 17 pre conversion drying 6 19 PTH amino acid solubilization 6 20 sample transfer and injection 6 20 sample volume 6 20 sensor functions 6 7 Table of guidelines 7 11 oxidation PTC peptide or PTC protein 2 3 P PITC see also R1 2 4 PITC delivery of 6 16 PL PVDF Peptide method 3 12 B 32 PL PVDF Protein method 3 12 B 32 polybrene sample extracting 6 19 Post Coupling Extraction 2 4 premix buffer 7 4 effecting positively charged PTH AAs 7 8 figure effect of adding 7 4 pressure passing leak test 5 6 procedure changing an active gradient 7 6 cleanup C 17 creatinga 5 11 electrical test 5 8 C 25 flask cycle for sample i
140. lick the PDF icon for the document to download it immediately e Fill in the information form if you have not previously done so then click Deliver Selected Documents Now to submit your order Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e mail delivery Getting Help D 5 Warranty Applied Biosystems Limited Warranty Statement Applied Biosystems warrants to the customer that for a period ending on the earlier of one year s from the completion of installation or 15 month s from the date of shipment to the customer the Warranty Period the Procise purchased by the customer the Instrument will be free from defects in material and workmanship and will perform in accordance with the minimum specifications set forth in set out in contained in the Instrument User s Manual and or the Instrument s Product Specification Sheet the Specifications During the Warranty Period if the Instrument s hardware becomes damaged or contaminated or if the Instrument otherwise fails to meet the Specifications Applied Biosystems will repair or replace the Instrument so that it meets the Specifications at Applied Biosystems expense However if the valves or reagent lines become damaged or contaminated or if the chemical performance of the Instrument otherwise deteriorates due to solvents and or reagents other than those supplied or expre
141. lready done so then click Ask Us RIGHT NOW You will receive an e mail reply to your question from one of our technical experts within 24 to 48 hours To Obtain Free 24 hour access to Applied Biosystems technical documents including MSDSs Documents on is available by fax or e mail or by download from our Web site Demand To order documents Then by index number a Access the Applied Biosystems Technical Support Web site at http www appliedbiosystems com techsupp b Click the Index link for the document type you want then find the document you want and record the index number c Use the index number when requesting documents following the procedures below by phone for fax delivery a From the U S or Canada call 1 800 487 6809 or from outside the U S and Canada call 1 858 712 0317 b Follow the voice instructions to order the documents you want Note There is a limit of five documents per request through the Internet for fax or e mail delivery a Access the Applied Biosystems Technical Support Web site at http www appliedbiosystems com techsupp b Under Resource Libraries click the type of document you want c Enter or select the requested information in the displayed form then click Search d In the displayed search results select a check box for the method of delivery for each document that matches your criteria then click Deliver Selected Documents Now or c
142. lt Cart Temp C 48 Cart Pulsed liquid None Cart Precycle Cart Precycle 48 Cart Precycle Cart Precycle 45 Cart Pulsed liquid None None Cart Begin 45 Cart Gas phase None None Cart Begin Gas phase 48 Cart PL PVDF Protein None None Cart Begin 48 Cart GP PVDF Protein None None Cart Begin Gas phase 48 Cart PL PVDF Peptide None None Cart Begin Flask Temp C 64 Flask Normal Flask Prep Pump Flask Blank Flask Standard 64 None Flask Standard 64 Flask Normal Flask Prep Pump Flask Blank Flask Standard 64 Flask Normal Flask Prep Pump Flask Blank Flask Standard 64 Flask Normal Flask Prep Pump Flask Blank Flask Standard 64 Flask Normal Flask Prep Pump Flask Blank Flask Standard 64 Flask Normal Flask Prep Pump Flask Blank Flask Standard Column Temp C 55 Fast Normal I Prep Pump Fast Normal I Fast Normal I 55 None Fast Normal I 55 Fast Normal I Prep Pump Fast Normal I Fast Normal I 55 Fast Normal I Prep Pump Fast Normal I Fast Normal I 55 Fast Normal I Prep Pump Fast Normal I Fast Normal I 55 Fast Normal I Prep Pump Fast Normal I Fast Normal I 55 Fast Normal I Prep Pump Fast Normal I Fast Normal I Method Name GP PVDF Peptide Run Gradient PTH Standards Inject Optimize Manual Injection Flask Optimize Default Default Default Default Default Default Cart Temp C 48 Cart GP PVDF
143. lt lt Z lt lt lt lt Z Z lt Z lt lt Z lt lt lt Z Z lt Z Z Z Z Z lt lt Z lt lt lt Z lt lt lt lt Z lt Number 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 Reagent X3 X3 S2 R4 R4 R4 Name Transfer to FC X3 Reserved Transfer to Flask gas Transfer to FC gas Select Cartridge A Select Cartridge B Select Cartridge C Select Cartridge D Ready Transfer to Flask Transfer Complete Pressurize Cart top Pressurize Cart bottom Dry Cart top Dry Cart bottom Dry Cart high top Dry Cart high bottom Flush Cart Reagent Block Flush Cart Solvent Block Flush Input Block Flush Output Block Flush Small Loop Cart Flush Large Loop Cart Flush Transfer Line Set Cart Temperature Wash Cart Reagent Block Wash Cart Solvent Block Wash Small Loop Cart Wash Large Loop Cart End Cartridge Select Cartridge Wait Wash Transfer Line S2 Reserved Del R4 Flask Load R4 Flask sm loop Load R4 Flask Ig loop Valves 4 7 17 18 19 20 34 35 36 37 39 15 23 17 18 19 20 34 35 36 37 38 45 15 23 17 18 19 20 34 35 36 37 39 23 17 18 19 20 15 10 34 35 36 37 15 23 17 18 19 20 34 35 36 37 40 15 10 17 18 19 20 34 35 36 37 16 15 23 17 18 19 20 34 35
144. lush Large Loop Flask 10 N 13 257 Wait 30 N 14 213 Dry Flask 150 N 15 236 Pre Conversion Dry 0 Y 16 218 Flush Large Loop Flask 10 N 17 153 Load R4 Flask Ig loop 20 N 18 213 Dry Flask 10 N 19 218 Flush Large Loop Flask 10 N 20 173 Load S4 Flask lg loop 15 N 21 218 Flush Large Loop Flask 15 N 22 257 Wait 540 N 23 237 Post Conversion Dry 0 Y 24 226 Load Position N 25 227 Prepare Pump 1 N 26 213 Dry Flask 450 N 27 173 Load SA Flask Ig loop 15 N 28 213 Dry Flask 10 N 29 218 Flush Large Loop Flask 10 N 30 173 Load S4 Flask Ig loop 15 N 31 213 Dry Flask 10 N 32 218 Flush Large Loop Flask 10 N 33 221 Flush Injector 30 N 34 257 Wait 5 N 35 221 Flush Injector 30 N 36 213 Dry Flask N 37 238 Concentrate Sample Y Cycle Method and Gradient Listings B 25 Step 38 39 40 41 42 43 44 45 46 47 48 49 50 Function 257 225 249 171 213 212 215 171 213 212 222 221 259 Flask Standard Total run time 36 07 Step O N OA R WD ND MO N N a do MO O ON O O O N Da sai WIN B 26 Cycle Method and Gradient Listings Function 258 234 171 213 215 151 213 215 218 257 163 213 236 218 153 213 218 173 218 257 237 226 227 Function Name Wait Load Injector Inject Pos Collect Data Del 54 Flask Dry Flask Bubble Flask Empty Flask Del 54 Flask Dry Flask Bubble Flask Flush Flask Injector Flush Injector End Function Name Begin Set as Standar
145. lush S1 Backflush S2 Backflush S3 Backflush X1 Backflush X19 Backflush X3 Cart Backflush X3 Flask Backflush R4 Backflush R5 Backflush S4 Backflush X2 Backflush X2g Flush Cart Reagent Block Flush Cart Solvent Block Select Cartridge A Transfer to Flask gas Dry Cart bottom Select Cartridge B Transfer to Flask gas Select Cartridge C Dry Cart top Select Cartridge D Dry Cart top End Cartridge Select Flush Input Block Flush Output Block Flush Small Loop Cart Flush Large Loop Cart Dry Flask h press Flush Small Loop Flask Flush Large Loop Flask Flush Flask Injector Inject Position Flush Flask Injector Empty Flask Global Time sec Time 50 0 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 120 120 120 120 120 60 60 60 60 120 60 60 60 60 60 N Z Z Zz Z Z z Z Zz Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Procedure Listings C 23 C 24 Procedure Listings Step 58 59 60 61 62 Function 303 305 303 305 259 Function Name Select Regulator Set Reg Setpoint 10th psi Select Regulator Set Reg Setpoint 10th psi End Time sec 5 35 8 30 0 Global Time N Z iz ZZ Electrical Test Procedure Electrical Test Step O N Asa 0 PM Function 258 311 312 313 314 315 316 259 Function Name Begin Test Valves Test Heaters Test Pressure Board Test 1
146. mable liquid and vapor lt may cause eye skin and respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves S3 n butyl chloride CHEMICAL HAZARD S3 n butyl chloride is a flammable liquid and vapor Exposure may cause central nervous system effects such as drowsiness dizziness headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves S4B 20 acetonitrile in water CHEMICAL HAZARD S4B 20 acetonitrile in water is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Sodium dodecyl sulfate SDS CHEMICAL HAZARD Sodium dodecyl sulfate SDS may cause an allergic respiratory reaction It is harmful if inhaled swallowed or absorbed through the skin Exposure causes eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Introduction and Safety 1 9 Sequence Analysis Chemistry Ove
147. mple loop load sensor detects fluid the injector valve is switched from the load to the inject position moving the sample into the HPLC solvent stream The HPLC begins running the programmed gradient and data collection is started The following steps are necessary in a flask cycle in order for a successful sample injection Steps Function Description Prepare Pump 227 Gradient information is downloaded to the pump from the Procise software After downloading is complete 30 60 seconds the pump will start pressurize and run at the initial gradient conditions Load Position 226 This function must precede the Load Injector step in order for the sample loop to be flushed before the sample in the flask is transferred into the sample loop Flush Injector 221 Flushes the sample loop from valve 44 Does not flush through the flask Load Injector 225 Activates the sample loop sensors Transfers sample from the flask into the HPLC sample loop The volume of sample transferred to the injector loop is determined by the size of the loop loads sent to the flask The standard volume of a large loop load of S4 is 60 pL Two loads to the flask provide a total sample volume of 120 uL Bubbling in the flask reduces the acetonitrile content of the sample in the flask reduces the sample volume and insures proper binding of the PTH amino acids to the HPLC column Injection Percentage The standard injection percentage fo
148. n PTH AA Separation The steps for optimizing the separation of specific PTH amino acids and chemistry at Installation artifacts are described in this section During installation the PTH amino acid separation is optimized for the PTH column supplied with your instrument The gradient used is stored in the Procise control software These conditions serve as the starting point To maintain optimum separation the gradient may require fine tuning as the column ages Positioning the The positively charged PTH AAs are Positively Charged Histidine PTH AAs sa Arginine Pyridylethyl cysteine Increasing the ionic strength of the mobile phase reduces the retention of the basic derivatives on the column Suggested elution positions for the basic derivatives are His between DMPTU and Ala Arg between Ser and Tyr and PECys before Pro For the majority of columns these elution positions can be obtained by using 20 mL of the Premix buffer concentrate per liter of solvent A3 and making minor gradient modification However increasing the buffer concentration can be a useful method to make Arg elute earlier than Ser and to make PECys elute earlier than Pro See Chemical Warnings Table on page 1 7 for warnings about A3 and premix buffer concentrate solutions If Then PECys is not a derivative of interest it is possible to position His after Ala and Arg after Tyr by using less Premix buffer Approximately 10 mL of
149. n and Safety 1 5 Reagents and Solvents Used on the System Table of Chemical All reagents and solvents supplied by Applied Biosystems are highly purified and Storage Conditions Sequencer tested to ensure optimal performance A list of reagents and solvents used with the standard sequencer cycles is given in Table 1 1 Table 1 1 Chemicals Used on the Procise Protein Sequencer Bottle Part Storage Position Chemical Contents Number Conditions R1 R1 5 phenylisothiocyanate PITC in heptane 400208 20 C a R2 R2B N methylpiperidine water methanol MeOH 401535 4 Ca R3 R3 Trifluoroacetic acid TFA neat 400003 RT R4 RAA 25 TFA in water with 0 01 dithiothreitol 400028 4 Ca DTT R5 PTH standard plus 400879 RT b R5 acetonitrile with 0 001 DTT 400315 S1 S1 n heptane not used 400079 RT S2 S2B ethyl acetate 400854 RT b S3 S3 n butyl chloride 400008 RT b S4 S4B 20 acetonitrile in water 400314 RT D x1 50 methanol water X2 Acetonitrile X3 Not used 20 Amino Acid PTH standard 400879 20 C a Biobrene Plus 400385 4 Ca B lactoglobulin 400979 4 Ca a Allow these items to reach room temperature before opening When these bottles are opened while still cold water can condense inside Check bottle caps for tightness after placing these bottles at either 4 C 2 8 C or 20 C 15 to 20 C b RT Room Temperature 15 20
150. n page 1 7 for warnings about argon gas and S4 solutions Startup procedures can be programmed from the Start Run view for the automatic run The startup procedure is executed immediately after sensor initialization Programming from Program the startup and shutdown procedures from the Start Run view Start Run View To program from the Start Run view Step Action 1 Select the Start Run view from the View pop up menu At the bottom of the screen are pop up menus for Startup and Shutdown procedures Click on the Startup pop up menu at the left bottom of the window and highlight the procedure to be used Select the Shutdown pop up menu at the right bottom of the window and highlight the procedure to be used Running A Startup To run a startup procedure from the Test view Procedure Step Action 1 Select the Test view from the View pop up menu 2 Click on the Startup button and select the Startup procedure 3 Click on the Start Test button to run the procedure Note Allow the procedure to run to completion 5 4 Tests and Procedures Idle Procedures About the Idle When the instrument is not in use oxygen diffuses slowly into the system and causes Procedure solvents and reagents to decompose and form by products The idle procedure is used to minimize sequencing problems due to chemical decomposition during an inactive period The idle procedure flushes
151. njection 6 20 flow list of C 26 idle 5 5 C 29 leak test 5 6 shutdown 5 7 C 45 startup 5 4 C 47 proline cleavage after 6 18 protein size and cleavage time 6 18 size and sequence cycle 6 15 PTH conversion 2 5 PTH standard 1 6 PTH amino acid PTH AAs optimizing aspartic and glutamic acid 7 9 Gin Thr and Gly 7 5 Met Val lle Lys and Lys Leu 7 10 separation 7 8 to 7 11 Index 3 separating and analysis theory 2 6 solubilizing 2 5 6 20 PTH Standards method 3 13 B 33 Pulsed liquid method 3 11 B 32 PVDF membrane 6 16 pyridylethyl cysteine 7 9 R R1 5 PITC in n heptane characteristics of 2 3 P N 1 6 R2B N methylpiperidine water methanol characteristics of 2 3 P N 1 6 R3 TFA 2 4 P N 1 6 R4A 25 TFA in water aqueous TFA P N 1 6 R4A 25 TFA in water 2 5 R5 acetonitrile 2 5 reagents P Ns 1 6 using argon 2 3 retention time figure effect of premix 7 4 run table of components 3 2 Run Gradient method 3 13 B 33 S S1 P N 1 6 S2B ethyl acetate 2 4 P N 1 6 S3 1 chlorobutane P N 1 6 purpose of 2 4 S3 butyl chloride also 1 chlorobutane 2 4 S4B 20 acetonitrile water 2 5 P N 1 6 solubilizing with 2 5 safety 1 2 sample electroblotted 6 16 glass fiber disk 6 19 injection percentage 6 21 optimizing PVDF membrane bound 6 19 size and cleavage time 6 18 size and sequence cycle 6 15 volume 6 20 samples protein or peptide 2 2 sensor creating error message 6 8 detecting bubbles 6 9 flagging an err
152. no acids are Aspartic acid Glutamic acid If Then Asp needs to be separated from the DTT peak decrease the initial B If the initial B is reduced below 8 DMPTU will probably move under Glu The DTT peak elutes immediately after the negative dip of the injection artifact Add a gradient step at 0 4 minutes decreasing the initial B causes Glu to coelute with DMPTU add a gradient step at 0 4 minutes and set the B to 14 16 The initial B can then be lowered below 5 without losing the Glu DMPTU separation Note Decreasing the initial B may cause Asp and phenylthiourea PTU a reaction product of PITC and NH to coelute the gradient adjustments fail to separate Asp from the DTT peak add 100 uL of neat TFA per liter of Solvent A PTH Separation 7 9 If Then Asp needs to be separated from a Increase initial B Increasing the initial phenylthiourea PTU reaction product of B to 8 10 will usually separate the PITC and NH 3 Asp peak before PTU moving it towards the DTT peak The DTT peak elutes immediately after the negative dip of the injection artifact b Decrease the flow rate A decrease in flow rate from 325 mL min 300 mL min will move Asp away from PTU without significantly impacting any other separation The B at 18 minutes should be decreased by 1 to maintain optimum separation of lle Lys Glutamic Acid if Then
153. ns of the same cycle or different cycles needed to sequence a peptide protein sample A typical method may start with one cycle then perform many repetitions of another cycle Methods also include the appropriate starting temperatures for the cartridge flask and column as well as the gradient to be run by the HPLC Standard methods those supplied by Applied Biosystems are permanently stored in memory The user can also create new methods or edit existing methods Gradient A gradient is a pump program that defines flow rate and solvent composition changes over time See Chapter 4 Creating Functions Cycles and Methods for more information on functions steps cycles and methods 3 2 Valves Functions Cycles and Methods Valves About Valves Delivery Valves Table Designed for Delivery Gas and chemical deliveries are controlled by valves that open to create delivery pathways to a particular destination such as the reaction cartridge Each valve is assigned a number The valve diagram Figure 3 1 on page 3 4 displays the placement of each valve Valves are opened activated and closed deactivated electrically Delivery valves are built together into valve blocks The chemical delivery system is managed by seven valve blocks interconnected with Teflon tubing Types of Valve Blocks Valve Block Control Function Cartridge reagent block Controls delivery of reagents R1 R2 X1 liguid and g
154. nt perform the bottle change procedure for that bottle position 3 a From the Manual Control view select Function 217 Flush Large Loop or Function 218 Flush Small Loop from the flask function list b Activate the appropriate flush function for 20 seconds to make sure that the loop is clear of any liquid 4 a Activate the load function for the bottle and loop of choice For example select Function 75 to load the small loop with reagent or solvent from the X1 bottle position b Watch for the appearance of a check mark next to the reaction flow sensor field at the top of the screen c Note the elapsed time and add 5 10 seconds d Enter this load time in the cycle for this function e Select All Off before leaving the Manual Control view 5 From the Function view enter the load time in the global time field for that function Once the time for loading the large loop has been determined the same value can be used for loading the small loop 6 12 Chemical Optimization Rules to Apply to The following rules apply to using flask load functions in a custom cycle nai Load The loop must be flushed for at least 10 seconds before the first loading netions ke The loop must be flushed for at least 10 seconds between loadings The loop must be washed and flushed between loadings of multiple reagents Chemical Optimization 6 13 Optimizing Deliver to Cartridge Sensor Functions Cartridge
155. nts after transfer from the cartridge The flask should not be completely dry after the pre conversion dry step or loss of certain amino acid derivatives will result The post conversion dry step eliminates the 25 TFA used to convert the ATZ amino acids to PTH amino acids The flask must be completely dry after the post conversion dry step to avoid chromatographic disturbance Valves Functions Cycles and Methods 3 13 Creating Functions Cycles and Methods Overview About This Chapter This chapter describes how to modify or create cycles and methods A complete listing of the functions cycles and methods can be found in Appendix A and B In This Chapter This chapter contains the following topics Topic See Page Creating User Defined Functions 4 2 Global Times 4 3 Creating Cycles 4 4 Creating Methods 4 7 Creating Functions Cycles and Methods 4 1 Creating User Defined Functions Creating Special Functions 401 450 are allocated for user defined functions Functions IMPORTANT Functions cannot be created or modified when the sequencer is in use To create special functions Step Action 1 Select the Functions view from the View pop up menu 2 Select one of the user functions using the up down scroll button and highlight the function Highlight the Function Name field and type in the function name Move the cursor to the Valves Activated field Ente
156. o value The following error message will be observed if a bottle runs dry during a run or if there is a restriction to reagent or solvent flow in the delivery path dry wet dry wet dry wet 5000 0 0 0 0 0 If the dry wet values reported in the event log are as follows then the sensor is detecting very small bubbles in the solvent or reagent stream This is a result of the solvent or reagent degassing as it flows through the valve blocks and can usually be corrected by reducing the bottle pressure for the particular chemical dry wet dry wet dry wet 5000 58 1 47 2 53 Chemical Optimization 6 9 Optimizing Cartridge Load Functions Two Cartridge Load Two load loops are available for metering reagents to the cartridge Loops A large loop which loads a nominal 10 pL of any cartridge reagent A small loop which loads a nominal 5 uL of any cartridge reagent The standard instrument cycles use the large loop for loading the cartridge reagents Loop Usage Large loop Delivers a volume of reagent that wets but does not saturate a glass fiber sample disk in the microcartridge 9 mm disk size The large loop volume of reagent is appropriate for blot samples whether run in the blot cartridge or a microcartridge Small loop A small loop load of TFA may be preferable for small pieces of PVDF or for smaller volume reaction cartridges Determining Note Whenever the delivery pressure for
157. o washout but does not require any other special considerations Typically there is no danger of over drying the sample at this point Chemical Optimization 6 17 Cleavage About the Cleavage Process Pulsed liquid Cleavage Gas phase Cleavage Drying After Cleavage 6 18 Chemical Optimization Cleavage whether pulsed liquid or gas phase is the TFA catalyzed process of removing the PTC amino acid from the amino terminal end of the sample Under strongly acidic conditions the peptide chain is cleaved at the peptide bond nearest to the PTC amino acid derivative resulting in the release of an ATZ amino acid The cleavage is not a hydrolytic process so ideally the sample should be as free of water as possible to minimize non specific hydrolytic cleavage of the peptide chain Pulsed liquid cleavage is performed by delivering a small aliquot of TFA to the cartridge on a stream of argon and sealing off the reaction chamber by closing the valves into and out of the cartridges to allow the cleavage to take place Pulsed liquid cleavage proceeds faster than gas phase cleavage The standard pulsed liquid cleavage time is 300 seconds at 48 C Certain samples may benefit by varying the cleavage conditions For example very large protein samples may sequence better using a shorter cleavage time to minimize amino acid background generated from non specific cleavage of certain peptide bonds Cleavage of the peptide bond after certain amino acids
158. omatography however the percentage of acetonitrile must be reduced before sample injection To reduce acetonitrile content two Concentrate Sample steps are included in all flask cycles The duration of these steps determines the percentage of sample injected Therefore the duration of the Concentrate Sample steps must be optimized so that the correct amount of sample is injected Optimize the injector percentage if a large number of Sample Loop Full error messages are recorded in the event log Two procedures are provided here one to set up the protein sequencer for optimization and the second to perform optimization To set up the protein sequencer for optimization Step Action 1 Scroll to the Functions dialog box 2 Ensure that the global time for the Concentrate Sample step step 238 is 100 sec 3 Place a mark 1 in from the hexagonal tip of the 5 16 in bushing on the yellow tubing connected to valve block port 42 4 Scroll to the Start Run dialog box E PROCISE Procise 1 0 File Edit Sequencer Help Idle Start Run v k Pause Later Run E Order ist w Cartridge File Name nos injector optote Cycles 30 2 Cycles Method Methoc Status Idle M Collect Data Sample 40 0 pmol Stdl40 0 pmol Startup None v Shutdown None v Start Run LZ 5 Set up a run with the following conditions Parameter Setting Cartridge A 1st Filename Your choice Number of cycl
159. on 258 266 303 47 49 260 48 44 136 144 136 259 Bottle Change for Total run time 2 55 S1 Leak C 8 Procedure Listings Step AN O Asa WD gt A N o Function 258 266 303 304 47 49 260 305 310 257 308 257 Function Name Restore Reg Setpoint Flush R5 Del R5 Waste Flush Large Loop Flask Wash Large Loop Flask Flush Large Loop Flask End Function Name Begin Set for Bottle S1 Select Regulator Vent S1 Backflush S1 Pause for Bottle Change Flush S1 Del S1 Waste Flush Cart Solvent Block Wash Cart Solvent Block Flush Cart Solvent Block End Function Name Begin Set for Bottle S1 Select Regulator Save Regulator Setpoint Vent S1 Backflush S1 Pause for Bottle Change Set Reg Setpoint 10th psi Set Tolerance 100th psi Wait Close Pressure Valve Wait Time sec 0 10 10 10 10 20 0 Time sec 0 0 4 10 20 0 15 10 10 5 20 0 Time sec 0 0 4 0 10 20 0 38 20 40 0 15 Global Time N Z ZIZ Z Z Z Global Time N Z Z 22 2 Z Z ZZ Z Z Global Time N Z ZZ Z SZZ sl ZIZ Z Zz Step 13 14 15 16 17 18 19 20 21 22 23 24 Function 307 317 310 257 318 309 48 44 136 144 136 259 Bottle Change for S2 Total run time 1 15 Step O O N OA R 0 PN Function 258 267 303 57 59 260 58 54 136 259 Bottle Change for Total run time 2 40 S2 Leak Step O N O WG FWD Fun
160. ons of the Edman chemistry The conditioning process reduces contaminants and improves the efficiency of subsequent sample sequencing Another method is optimized for samples bound to polyvinylidine difluoride PVDF membrane A specially designed reaction chamber the Blott cartridge is used for these membrane bound samples The Blott cartridge is a vertical cross flow reactor that facilitates optimization of the cartridge chemistry 2 2 Sequence Analysis Chemistry Edman Degradation The Coupling Reaction Coupling with R1 Coupling with R2B S QE HN RI N CHg RI O O L ones NH NH R N MePip R2 H2O R O EEE O Heat Protein PTC Protein PITC R1 R1 5 PITC in n heptane is a sweet smelling volatile liquid In the presence of base PITC R1 reacts quantitatively with the free amino groups of a peptide or protein including side chain amino groups to form the phenylthiocarbamyl derivative PTC derivative In standard sequencer cycles R1 is loaded and delivered to the cartridge on a stream of argon to wet the glass fiber filter Three separate R1 deliveries are alternated with vapor deliveries of base R2B These repeated R1 deliveries increase the reagent protein contact and improve the reaction yield After each R1 delivery argon removes the non reactive solvent n heptane R2B N methylpiperidine water methanol is a strongly basic amine identifiable in higher concentrations by its strong ammonia
161. or for cleavage Includes pre wetting of the membrane with methanol water from X1 bottle prior to coupling and an additional extraction of the membrane after cleavage Three ATZ extractions two with butyl chloride one with ethyl acetate Valves Functions Cycles and Methods 3 9 Table 3 7 Standard Cartridge Cycles continued Cycle Description Cart GP PVDF Peptide Edman chemistry cycle for sequencing peptide samples on PVDF membrane Delivers TFA vapor for cleavage Three ATZ extractions two with butyl chloride one with ethyl acetate Standard Flask There are five standard flask chemistry cycles A complete flask cycle listing can be Cycles found in Flask Cycle List on page B 25 Table 3 8 Standard Flask Cycles Cycle Description Flask Blank Performs conversion chemistry and reconstitution without any sample or standard Starts HPLC and transfers flask contents to the sample loop for analysis Flask Standard Performs conversion chemistry and reconstitution with PTH amino acid standard mixture in the flask Starts HPLC and transfers flask contents to the sample loop for analysis Flask Normal For converting the ATZ amino acid transferred from the cartridge to a PTH amino acid Starts HPLC and transfers flask contents to the sample loop for analysis Flask Prep Pump Downloads instructions to the HPLC pump to run at 50 B while the flask blank is being prep
162. or message 6 8 function of 6 7 Init 5 5 optimizing deliver to cartridge 6 14 Index 4 test sensor and delivery C 27 types of 6 7 serine 7 9 shutdown describing Post Run Valve Block Wash X3 5 7 overview 5 7 short term procedure to C 45 solubilizing PTH amino acids 6 20 Solvent A3 3 5 tetrahydrofuran water 7 3 solvents P Ns 1 6 removing by products 2 4 solvent A3 7 3 solvent B2 7 3 solvent B2 12 isopropanol in acetonitrile 7 3 using argon 2 3 standard 20 AA PTH 1 6 B lactoglobulin PN 1 6 step defining a 3 2 system clean out X3 describing 5 8 system flush argon describing 5 8 T technical support D 1 to D 5 e mail address D 1 Internet address D 5 regional sales offices D 3 telephone fax U S D 2 test procedure 5 2 to 5 3 creating a 5 11 defining electrical 5 8 electrical test C 25 flow tests 5 3 leak test 5 6 leak tests C 32 optical sensors 5 3 sensor and delivery step functions C 27 tests and procedures 5 2 to 5 11 TFA see also R3 2 4 thiourea formation of 2 4 threonine separating 7 5 transfer optimizing 6 20 V valine separating from methione 7 10 valve 3 3 to 3 5 bottle vent valve 3 5 defining a 3 2 status function table 3 5 three way valve 3 5 valve block diagram of 3 4 table types of 3 3 valve block leak test 5 6 W Warnings chemical summary table 1 7 wash post coupling 6 17 waste leaktest C 44 WWW address Applied Biosystems D 1 Documents on Demand D 5
163. own Procedute List ass sveise Ne 99 RA ie ele wed C 45 Startup Procedure see aaa il e i a C 47 Getting Help Technical Supports tirano E pe a D 1 Warranty Applied Biosystems Limited Warranty Statement E 1 Index Introduction and Safety Overview About This Chapter This chapter describes the user attention words used throughout this manual and safety issues pertaining to Applied Biosystems instrumentation In This Chapter This chapter contains the following topics Topic See Page Safety 1 2 Reagents and Solvents Used on the System 1 6 Introduction and Safety 1 1 Safety Documentation User Attention Words Chemical Hazard Warning Chemical Waste Hazard Warning 1 2 Introduction and Safety Five user attention words appear in the text of all Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation Fe NU EN Cautions the user that a potentially hazardous situation could occur causing injury to the user or damage to the instrument if this information is ignored PNA Warns the user that serious physical injury or death to the user or other persons could result if these precautions are not taken L Aa Indicates an imminently hazardous situation that if not avoided will res
164. ptimizing Flask Load Functions 6 12 Optimizing Deliver to Cartridge Sensor Functions 6 14 Chemistry Optimization 2 6 15 Couplng su serberne ener Te and dt ka 6 16 OE EEE EEE ER Gets 6 18 PTH Separation OVervIeW i Frisia adora ie tene ka 7 1 Amino Acid Table sne see ee ea 7 2 HPLC DOMNENUS ciel SEN SR SERRA 7 3 Gradient and Programming 7 5 Optimizing the Chromatography 7 7 Optimizing the PTH AA Separation 7 8 Function Listing Overview Loire RAI Eek Bg ee EA EOL I Se IRENE A 1 Function List Fable sacri iii i eli noe ee ot d ai A 2 Cycle Method and Gradient Listings OVEIVICW RE Ata B 1 Cartridge Cycle List cui ii la heste nes ka B 2 Fl sk Cycle E coil aa ei iii AE e a B 25 Method EIA oaks Wards WG SAT ER EE SE B 32 Gradient List us bici s re icon B 34 Procedure Listings OVEEVIEW pak Seles eb hake ole sveaerne eat C 1 Bottle Change Procedure List iii pae aaa C 2 Cleanup Procedure List 5 g e gar 5 ii hl hee Lei i C 17 Electrical Test Procedure 255 tilllate ani lea C 25 Flow Procedure List nu rr LEVELS Aeree evita C 26 TdleProcedure i 6 iiiciielaliceiale dit SE C 29 Init Sensor Procedure C 30 Leak Procedure TAS iii C 32 Shutd
165. r each extraction solvent is delivered to the cartridge outlet sensor allowed to incubate with the sample for 10 seconds and then transferred to the flask with argon S2B which is more polar than S3 improves the recovery of polar residues particularly histidine arginine aspartic acid and glutamic acid Using S3 for the first extraction reduces the possibility of polybrene sample washout The argon delivery after each extraction must be long enough to transfer the contents of the cartridge to the flask and dry the cartridge outlet sensor If there are still droplets of liquid at the outlet sensor solvent delivery may be cut short Samples applied to PVDF membrane whether sequenced using gas or liquid cleavage are extracted in the same way PVDF membrane blot cycles use three extractions the first with S3 butyl chloride the second with S2B ethyl acetate and the third with S3 For each extraction solvent is delivered to the cartridge outlet sensor allowed to incubate with the sample for 10 seconds and then transferred to the flask with argon As with samples on glass fiber extraction with S2B improves the recovery of polar residues The argon delivery after each extraction must be long enough to transfer the contents of the cartridge to the flask and dry the cartridge outlet sensor If there are still droplets of liquid at the outlet sensor solvent delivery may be cut short The ATZ amino acid is extracted from the cartridge and t
166. r the Procise Protein Sequencer is 70 to 80 This percentage of injection was selected to provide consistent fluid detection at both the Sample Loop Load and Sample Loop Full sensors The percentage of sample injected can be increased by lengthening the global time setting for the Concentrate Sample step function 238 in order to reduce the total sample volume At greater than 80 injection intermittent failures to detect fluid may be reported to the Event Log by the Sample Loop Full Sensor The reduced sample volume may not be enough to consistently provide a wet reading at the Sample Loop Full Sensor Chemical Optimization 6 21 PTH Separation Overview About This Chapter This chapter describes the procedures used for the Procise liquid chromatography HPLC system Instructions for preparing solvents and optimizing the separation of PTH amino acids are in this section Routine operations of the Procise HPLC system are controlled by Procise control software For complete descriptions of the menus used to control the pumps refer to the Model 140C Micro gradient Delivery System User s Manual In This Chapter This chapter contains the following topics Topic See Page Amino Acid Table 7 2 HPLC Solvents 7 3 Gradient and Programming 7 5 Optimizing the Chromatography 7 7 Optimizing the PTH AA Separation 7 8 PTH Separation 7 1 Amino Acid Table Abbreviations Table 7 2 PTH Separation Table 7 1 Amino A
167. r the valve numbers to be activated for the function Enter a space between each valve number IMPORTANT The number of valves that can be activated per function is limited by the following rules Rule 1 For valves 1 23 34 40 45 46 and 63 a maximum of 8 valves can be activated per function For valves 24 33 41 44 and 47 62 a maximum of 6 valves can be activated per function Rule 2 A maximum of 14 valves total 8 cartridge and 6 flask can be activated simultaneously Repeat steps 2 through 5 for all functions you create Pull down the File menu from the upper menu bar Select Save Function to save all new functions 4 2 Creating Functions Cycles and Methods Global Times About Global Time Most functions of the Procise Protein Sequencer can be run with a global time setting The global time is set from the Function view For any cycle in which the Global time box is checked for a particular function the Global time value from the Function view will be used as the time for that function In this way the duration of a function can be modified wherever it is used in multiple cycles by making a single change in the Global time field Standard cycles use global time only for the cartridge function Load X1 and the flask functions Pre Conversion Dry Post Conversion Dry and Concentrate Sample Setting Global Time IMPORTANT Global times cannot be set when the sequencer is in use To set the global time
168. ragraphs The goal of a protein sequencing run on an unknown sample is to unambiguously identify as many amino acids as possible using the least amount of sample The length of a protein sequence that can be determined is limited by the chemical efficiency of the Edman degradation as well as the purity amount and molecular weight of the sample to be sequenced Because the chemical efficiency is less than 100 the amount of sample you can sequence decreases slightly with each successive degradation cycle With the exception of the initial coupling the reaction of PITC with the amino terminus or termini proceeds nearly quantitatively The particular amino acid being reacted or the local structure of the peptide chain has little effect on the efficiency of the coupling reaction The cleavage reaction requires strong acid however so a balance must be struck between complete cleavage of the ATZ amino acid from the peptide and unwanted acid cleavage at other sites along the peptide chain Because of this balance cleavage efficiency is affected by the amino acid derivative being cleaved as well as the next amino acid in the chain Incomplete cleavage of the ATZ amino acid is referred to as lag and the remaining uncleaved portion of the current N terminal amino acid will appear in the chromatogram for the next cycle along with the next amino acid Lag increases with each cycle in a sequencing run and depending on the particular amino acids in th
169. ransferred to the flask for conversion into the more stable PTH amino acid derivative In preparation for the transfer of the ATZ amino acid to the flask a small volume of 20 acetonitrile S4 is delivered to the flask The presence of the S4 in the flask reduces the modification of certain amino acid residues in particular serine and threonine During transfer and immediately after transfer the liquid in the flask is bubbled to evaporate the S3 and S2B transferred from the cartridge and to reduce the volume of the sample prior to the addition of R4 to the flask for conversion At this point in the conversion cycle the sample should not be completely dried in the flask but reduced to a volume of 10 20 uL Completely drying the sample before conversion will cause reduced recovery of labile residues particularly serine and threonine To simplify optimization of this drying step function 236 Pre conversion Dry has a global time setting Changing the global time setting in the Function view will adjust the length of this step in any cycles in which it is used See Setting Global Time on page 4 3 for instructions on modifying a global time setting Conversion of the ATZ amino acid into a PTH amino acid takes place in aqueous acid medium A large loop load of R4 is added to the flask and allowed to incubate with the sample for approximately 9 minutes It is possible to use a small load of R4 for the Chemical Optimization 6 19 Post Convers
170. ress producing effects such as fatigue pain and strain To minimize these effects on your back legs eyes and upper extremities neck shoulder arms wrists hands and fingers design your workstation to promote neutral or relaxed working positions This includes working in an environment where heating air conditioning ventilation and lighting are set correctly See the guidelines below MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by the following potential risk factors which include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors Use a seating position that provides the optimum combination of comfort accessibility to the keyboard and freedom from fatigue causing stresses and pressures The bulk of the persons weight should be supported by the buttocks not the thighs Feet should be flat on the floor and the weight of the legs should be supported by the floor not the thighs Lumbar support should be provided to maintain the proper concave curve of the spine Place the keyboard on a surface that provides The proper height to position the forearms horizontally and upper arms vertically Support for the forearms and hands to avoid muscle fatigue in the upper arms Position the viewing screen to the height that allows normal body and head postur
171. rimarily eliminates the water absorbed by the polybrene during the coupling reaction Some of the reaction chemicals will also be reduced during this step but the subsequent wash will remove the bulk of the chemistry by products This drying can be extended with no concern for the loss of recovery of any residues The goal is to eliminate as much water as possible before the wash and cleavage to prevent sample washout and hydrolysis of the peptide chain during the cleavage The purpose of the post coupling wash is to remove as much of the coupling reagents and reagent by products as possible before the cleavage reaction begins The washing is done with a combination of the solvents S2B and S3 The washing scheme of short deliveries of solvent alternating with brief cartridge wait steps reduces the likelihood of sample washout and provides the best efficiency of washing with minimal solvent consumption The first delivery of solvent to the cartridge is S3 the less polar of the solvents to reduce the possibility of sample being washed out of the reaction cartridge Increasing the volume of solvent used for post coupling washing will reduce the chemistry background but may increase the loss of sample from the cartridge due to washout particularly if short hydrophobic peptides are being sequenced In particular lengthy S2B washings will aggravate sample washout Drying after the post coupling wash should completely dry the sample to prevent loss t
172. rs TFA vapor to the cartridge for cleavage of the ATZ amino acid A few microliters of BiobrenePlus solution diluted 1 10 with methanol should be applied to the sample membrane before sequencing 3 12 Valves Functions Cycles and Methods Table 3 9 Standard Methods continued Method Used to Run Gradient evaluate the performance and integrity of the HPLC system and the UV detector The Run Gradient method activates the HPLC system and starts data collection without activating the Rheodyne injector valve PTH Standards verify the performance and integrity of the HPLC system UV detector HPLC column Rheodyne injector valve and conversion flask Conversion reagents The PTH standards method performs a complete conversion cycle on the PTH standard mixture in the conversion flask activates the HPLC system and injects the standard onto the HPLC column for separation and analysis Inject Optimize adjust the volume of the sample in the flask after reconstitution and prior to loading into the HPLC sample loop Manual Injection verify the performance and integrity of the HPLC system UV detector HPLC column Rheodyne injector valve excluding the conversion flask Conversion reagents Flask Optimize optimize the flask drying steps pre conversion dry and post conversion dry The pre conversion dry step concentrates the sample and evaporates the extraction solve
173. rview About This Chapter This chapter describes the Edman chemistry used by the Procise Protein Sequencing System A description of the chemicals their stability and storage requirements is included here The analysis of PTH amino acids by reversed phase HPLC is also outlined In This Chapter This chapter contains the following topics Topic See Page Introduction 2 2 Edman Degradation 2 3 Sequence Analysis Chemistry 2 1 Introduction About the Chemical The Procise Protein Sequencer automates a chemical process that removes and Process analyzes amino acid residues from protein and peptide chains of various types and lengths This process is derived from the technique developed by Pehr Edman for the sequential degradation of proteins and peptides Edman degradation relies on the specific reactivity of a protein s N terminal amino acid and the selective removal of the derivatized amino acid from the protein while leaving the rest of the molecule intact Each cycle of the degradation sequentially removes an amino acid from the amino terminal end of the protein or peptide sample This cyclic process provides the primary structure Two Sequencing The Procise Protein Sequencer offers a variety of sequencing methods One method Methods is optimized for protein or peptide samples applied to a glass fiber filter previously treated with BioBrene Plus and then cycled through a conditioning process of one or more repetiti
174. s been determined the same value can be used for loading the small loop 6 10 Chemical Optimization Rules to Apply to The following rules apply to using cartridge load functions in a custom cycle NE Load The loop must be flushed for at least 10 seconds before the first loading netions NE CE The loop must be flushed for at least 5 seconds between loadings The loop must be washed and flushed between loadings of multiple reagents Chemical Optimization 6 11 Optimizing Flask Load Functions Two Flask Load There are two load loops available for the flask Unlike the cartridge load loops the Loops Volume of any particular reagent or solvent loaded depends on the position of that chemical on the valve block Reagent Solvent Small Loop HL Large Loop pL 54 25 60 X3 20 55 X2 15 50 R4 10 45 R5 5 40 Determining Flask IMPORTANT Whenever the delivery pressure for a reagent is changed load times must be Load Function Time changed as well IMPORTANT If the protein sequencer has not been run since the last cold start use the Test view to select and run the Init Sensor procedure Then allow the procedure to run to completion To determine the amount of time necessary for a flask load function Step Action 1 From the Pressures and Temperatures view set the desired delivery pressure for the bottle position to be used 2 If the reagent or solvent is not loaded on the instrume
175. ss of failure box is unchecked a dialogue box will appear on the screen to bring attention to each failure as it arises In this mode it is necessary to manually resume the test s after each reported failure In both cases all test results are reported in the Event Log 4 Use the Test pop up menu and highlight the test procedure to be used Multiple procedures can be selected by holding down the shift key 5 Click on the Start Test button to run the selected test The Stop Test button and the status field become active indicating that the test or procedure is running 6 Press the Stop Test button to interrupt the procedure IMPORTANT Whenever possible allow all Tests and Procedures to run to completion Certain procedures adjust the pressure settings for reagent solvent or gas delivery If the test or procedure must be interrupted go to the Pressures and Temperatures view and make sure that all pressures are set to a value other than the operating value If pressures have been set to Zero click on the Default button to restore original pressures 5 2 Tests and Procedures Flow Test Procedures Test Availability There are seven flow test procedures for the Procise system The procedure listed in Sensor and Delivery Test Procedure Table 5 1 can be performed by users the remaining procedures are used only during instrument manufacture and are not useful with the standard sequencing reagents
176. ssly recommended by Applied Biosystems Applied Biosystems will return the Instrument to Specification at the customer s request and at the customer s expense After this service is performed coverage of the parts repaired or replaced will be restored thereafter for the remainder of the original Warranty Period This Warranty does not extend to any Instrument or part which has been a the subject of an accident misuse or neglect b modified or repaired by a party other than Applied Biosystems or c used in a manner not in accordance with the instructions contained in the Instrument User s Manual This Warranty does not cover the customer installable accessories or customer installable consumable parts for the Instrument that are listed in the Instrument User s Manual Those items are covered by their own warranties Applied Biosystems obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct those failures of the Instrument to meet the Specifications of which Applied Biosystems is notified prior to expiration of the Warranty Period All repairs and replacements under this Warranty will be performed by Applied Biosystems on site at the Customer s location at Applied Biosystems sole expense No agent employee or representative of Applied Biosystems has any authority to bind Applied Biosystems to any affirmation representation or warranty concerning the Instrument that is not contain
177. ssure Open Valves 7 11 15 16 Set Tolerance 100th psi Compare Pressures 10th psi Restore Reg Setpoint Open Valves 7 11 15 16 End Time sec 10 38 30 10 10 10 Time sec 0 5 0 5 38 20 15 1 10 38 30 10 10 10 Global Time N Z 2 222 Z Z Z Z ZZ Global Time Z Z ZZ en LL Le 2 Z ZZZ ZZ AS Z Flask Input Test Total run time 1 05 Flask Leak Test Step AN OA R 0 PM a a dA dA A A dd co O N O WG RARA OON SOI Function 258 303 304 305 310 294 308 294 307 317 294 310 318 218 310 307 309 218 259 Total run time 2 00 Step AN DU FWD 11 12 13 14 15 Function 258 303 304 305 310 295 308 295 307 317 295 310 318 296 310 Function Name Begin Select Regulator Save Regulator Setpoint Set Reg Setpoint 10th psi Set Tolerance 100th psi Open Valve 24 Close Pressure Valve Open Valve 24 Compare Pressures 10th psi Save Regulator Pressure Open Valve 24 Set Tolerance 100th psi Compare Saved Pressure Flush Large Loop Flask Set Tolerance 100th psi Compare Pressures 10th psi Restore Reg Setpoint Flush Large Loop Flask End Function Name Begin Select Regulator Save Regulator Setpoint Set Reg Setpoint 10th psi Set Tolerance 100th psi Open Valves 24 32 Close Pressure Valve Open Valves 24 32 Compare Pressures 10th psi Save Regulator Pressure Open Valves 24 32 Set Tolerance 100th psi Compare
178. t top 15 N 88 148 Cartridge Wait 5 N 89 131 Dry Cart top 120 N 90 259 End 0 N Cart GP PVDF Total run time 41 05 Peptide Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 137 Flush Input Block 5 N 3 11 Del R2g Cart top 30 N 4 140 Flush Large Loop Cart 10 N 5 6 Load R1 Cart Ig loop 20 N 6 131 Dry Cart top 30 N 7 140 Flush Large Loop Cart 5 N Cycle Method and Gradient Listings B 19 Step 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 B 20 Cycle Method and Gradient Listings Function 135 11 132 140 6 131 140 135 11 132 140 6 131 140 135 11 146 111 140 137 136 132 142 63 148 61 148 51 148 51 148 131 53 148 51 148 51 148 61 148 131 Function Name Flush Cart Reagent Block Del R2g Cart top Dry Cart bottom Flush Large Loop Cart Load R1 Cart Ig loop Dry Cart top Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Dry Cart bottom Flush Large Loop Cart Load R1 Cart Ig loop Dry Cart top Flush Large Loop Cart Flush Cart Reagent Block Del R2g Cart top Wash Large Loop Cart Wash Input Block S3 Flush Large Loop Cart Flush Input Block Flush Cart Solvent Block Dry Cart bottom Set Cart Temperature Del S3 Cart sensor Cartridge Wait Del S3 Cart top Cartridge Wait Del S2 Cart top
179. t Temperature Del S3 Cart sensor Cartridge Wait Del S3 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Dry Cart top Del S2 Cart sensor Cartridge Wait Del S2 Cart top Cartridge Wait Del S2 Cart top Cartridge Wait Del S3 Cart top Cartridge Wait Dry Cart top Del R3g Waste Del R3g Cart top Wash Cart Reagent Block Flush Cart Reagent Block Wash Cart Solvent Block Flush Cart Solvent Block Dry Cart top Set Cart Temperature Ready Transfer to Flask Flush Transfer Line Del S3 Cart sensor Time sec 20 30 5 5 170 10 10 15 20 30 60 48 15 10 10 10 5 720 15 30 10 30 40 48 Global Time N Z Zz Z Ziz Z Z Zz RZ ZA ZZ ZZ Z Z Z Zz Z ZZ ZZZ LL LL Z Z Z Z E E O Global Step Function Function Name Time sec Time 64 148 Cartridge Wait 10 N 65 121 Transfer to Flask gas 45 N 66 141 Flush Transfer Line 5 N 67 53 Del S2 Cart sensor 15 N 68 148 Cartridge Wait 10 N 69 121 Transfer to Flask gas 45 N 70 141 Flush Transfer Line 5 N 71 63 Del S3 Cart sensor 15 N 72 148 Cartridge Wait 10 N 73 121 Transfer to Flask gas 45 N 74 128 Transfer Complete 0 N 75 131 Dry Cart top 60 N 76 61 Del S3 Cart top 15 N 77 148 Cartridge Wait 5 N 78 131 Dry Cart top 120 N 79 259 End 0 N Cart PL PVDE Total run time 33 45 Peptide Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 137 Flush Input Block 5 N
180. t the copied cycle Cycle y Step Action 1 To delete a row highlight the row to be deleted and click the Delete Row button 2 To insert a row select the function to be inserted from the function list on the left side of the screen The function can be selected by using the scroll down button or by typing the function number at the top corner of the function list a Highlight the row immediately before the intended insertion point b To enter the function run time click on the global box to turn the global time off Type the function time in seconds in the value box Saving the Edited To save the edited cycle Cycle Step Action 1 Pull down the File menu from the upper menu bar 2 Select Save Cycle Procedures IMPORTANT The Ready to Transfer step in a Cartridge cycle synchronizes with the Ready to Receive step in a Flask Cycle The cartridge cycle must have Ready to Transfer and Transfer Complete steps to transfer sample to the flask The maximum number of steps allowed per cycle is 100 Every cycle needs a Begin step and an End step 4 4 Creating Functions Cycles and Methods Copying Existing To copy the Procise standard cartridge cycle Flask Cycles Step Action 1 Use the View menu to select the Cycles and Procedures View 2 Use the upper screen menu to select Flask Cycle for cycle type 3 Use the lower screen menu to sele
181. ter cleavage must balance between recovery of particular residues especially the basic residues whose recoveries are drastically reduced if the sample is overdried and excessive washout if the sample is still too acidic when the extractions are done After cleavage care should be used to prevent overdrying of the sample which could result in poor extraction of charged residues and dehydration of labile residues In the standard pulsed liquid cycles 40 seconds of drying time is used Incomplete drying may cause lowered repetitive yields due to sample washout If sample washout is a greater concern than recovery of positively charged residues the drying after cleavage can be extended ATZ Extraction and Transfer Liquid Samples PVDF Membrane bound Samples Flask Chemistry Pre Conversion Drying Conversion After the cleavage of the ATZ amino acid from the peptide chain is complete and the sample dried the ATZ amino acid is extracted from the cartridge and transferred to the flask so that the coupling of the new amino terminus of the sample can begin The best method for the ATZ extraction is slightly different for the various sample types Samples applied to glass fiber disks with polybrene whether sequenced using gas or liquid cleavage are extracted in the same way Each glass fiber cycle has two ATZ extractions the first extraction is done with S3 butyl chloride the second with S2B ethyl acetate and the third with S3 Fo
182. the edited procedure Procedure Step Action 1 Pull down the File menu from the upper menu bar 2 Select Save Cycle Procedure IMPORTANT The maximum number of steps allowed per cycle is 100 Every cycle needs a Begin step and End step at the beginning and end of the cycle respectively Tests and Procedures 5 11 Chemical Optimization Overview About This Chapter This chapter provides guidelines for the development and optimization of functions cycles and methods for the Procise Protein Sequencer It includes explanations of the optimization of loop loadings deliveries and washing and drying steps for both the cartridge and the flask In This Chapter This chapter contains the following topics Topic See Page Optimizing the Injector 6 2 Optimizing Flask Dry Times 6 4 Optimizing Sensor Functions 6 7 Sensors and the Event Log 6 8 Optimizing Cartridge Load Functions 6 10 Optimizing Flask Load Functions 6 12 Optimizing Deliver to Cartridge Sensor Functions 6 14 Chemistry Optimization 6 15 Coupling 6 16 Cleavage 6 18 Chemical Optimization 6 1 Optimizing the Injector Reconstituting the Sample Reducing Acetonitrile Content Setting Up the Protein Sequencer 6 2 Chemical Optimization During flask cycles a sample is reconstituted in 10 acetonitrile S4C This ensures that hydrophobic amino acids will go into solution For consistent chr
183. ting a new 4 6 defininga 3 2 modifying a cartridge cycle 4 4 modifying a flask cycle 4 5 table of standard cartridge 3 9 standard flask 3 10 cycles flask optimizing flask dry times 6 4 to 6 6 D delivery line back flush describing 5 8 Documents on Demand D 5 DPU resolving from PTH Trp 7 3 drying after cleavage 6 18 after coupling 6 17 optimizing conversion 6 19 post conversion 6 20 pre conversion optimizing 6 19 E Edman chemistry optimizing 6 15 Edman degradation 2 2 2 3 cleavage 2 4 coupling 2 3 Rx diagram 2 3 electroblotting of sample 6 16 e mail address for technical support D 1 end step function 259 3 6 error detecting no flow 6 9 error posting of 6 8 ethyl acetate see also S2B 2 4 event log using the 6 8 extraction S2B and S3 2 4 using liquid samples 6 19 F Fast Precycle method 3 11 B 32 Filter Precycle method 3 11 B 32 flask cycles optimizing flask dry times 6 4 to 6 6 optimizing load functions 6 12 flask cycle procedures B 25 to B 30 blank B 25 flask optimize flsk B 30 flask standard B 26 manual injection B 30 normal B 27 prep pump B 29 run gradient B 28 flask leak test 5 6 flask load guidelines 6 13 Flask Optimize method 3 13 B 33 Index 2 flask optimizing chemistry 6 19 flow detecting no 6 9 function 3 6 to 3 8 begin step 3 6 cartridge load guidelines 6 11 cartridge loading time 6 10 creating special 4 2 cycle synchronizing 3 6 defininga 3 2 determining flask load time 6 12
184. tion 1 800 762 4001 then press 1 for PCR 2 for the 7700 or 5700 6 for the 6700 or dial 1 800 831 6844 then press 5 1 240 453 4613 Voyager MALDI TOF Biospectrometry and Mariner ESI TOF Mass Spectrometry Workstations 1 800 899 5858 then press 13 1 508 383 7855 Biochromatography BioCAD Workstations and Poros Perfusion Chromatography Products 1 800 899 5858 then press 14 1 508 383 7855 Expedite Nucleic acid Synthesis Systems 1 800 899 5858 then press 15 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 then press 15 1 508 383 7855 PNA Custom and Synthesis 1 800 899 5858 then press 15 1 508 383 7855 FMAT 8100 HTS System and Cytofluor 4000 Fluorescence Plate Reader 1 800 899 5858 then press 16 1 508 383 7855 Chemiluminescence Tropix 1 800 542 2369 U S only Or 1 781 271 0045 1 781 275 8581 Applied Biosystems MDS Sciex 1 800 952 4716 1 650 638 6223 Outside North America Region Telephone Dial Fax Dial Africa and the Middle East Africa English Speaking and West Asia Fairlands South Africa 27 11 478 0411 27 11 478 0349 South Africa Johannesburg 27 11 478 0411 27 11 478 0349 Middle Eastern Countries and North Africa Monza Italia 39 0 39 8389 481 39 0 39 8389 493
185. tivated opened for a fixed time as prescribed in a cycle or by the user in manual control mode and then deactivated at the end of the step The majority of functions operate in this manner The flowpath created by activating a valve controlling function can be traced using the valve diagram Figure 3 1 on page 3 4 Sensor functions are specialized valve controlling functions These functions are controlled by the target sensor rather than by a specific time setting When a function is activated the sensor begins looking for fluid When fluid is detected by the sensor the reagent or solvent delivery valve is turned off The time for the function continues to count down to 0 and the next step begins For a sensor function to perform correctly enough time must be allotted in the step for fluid to reach the sensor If fluid does not reach the sensor within the allotted time an error will be reported in the event log and the sequencer may be paused A group of functions are used to provide proper synchronization of the cartridge and flask cycles during sequencing Every cycle must have Begin Function 258 and End Function 259 steps in order to be valid The flask or the cartridge cycle will wait at the Begin step if necessary in order to keep the cycles synchronized The cycle timer will count up increment during this step if a cycle is synchronizing At the appropriate time the cycle will automatically proceed to the next step
186. top Wait Set Reg Setpoint 10th psi Set Tolerance 100th psi Pressurize Cart top Close Pressure Valve Pressurize Cart top Compare Pressures 10th psi Time sec 0 5 0 0 10 1 35 20 30 1 15 35 0 10 30 Time sec 0 5 0 0 10 1 35 20 30 1 15 35 Global Time Z ZZZ LANZO Z z ZZ Z Z Z ZZ Global Time N Z ZZ Z ZZ Zz Z Z Z Z Global Step Function Function Name Time sec Time 13 317 Save Regulator Pressure 0 N 14 310 Set Tolerance 100th psi 10 N 15 129 Pressurize Cart top 30 N 16 318 Compare Saved Pressure N 17 309 Restore Reg Setpoint N 18 131 Dry Cart top 10 N 19 147 End Cartridge Select N 20 259 End N Cartridge C Leak Total run time 1 36 Test Global Step Function Function Name Time sec Time 1 258 Begin 0 N 2 303 Select Regulator 5 N 3 304 Save Regulator Setpoint 0 N 4 125 Select Cartridge C 0 N 5 131 Dry Cart top 10 N 6 257 Wait 1 N 7 305 Set Reg Setpoint 10th psi 35 N 8 310 Set Tolerance 100th psi 20 N 9 129 Pressurize Cart top 30 N 10 308 Close Pressure Valve 1 N 11 129 Pressurize Cart top 15 N 12 307 Compare Pressures 10th psi 35 N 13 317 Save Regulator Pressure 0 N 14 310 Set Tolerance 100th psi 10 N 15 129 Pressurize Cart top 30 N 16 318 Compare Saved Pressure N 17 309 Restore Reg Setpoint N 18 131 Dry Cart top 10 N 19 147 End Cartridge Select N 20 259 End N Cartridge D Leak Total run time 1 36 Test Global Step Function Function Name Time
187. ult in death or serious injury FINZI CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with and inhalation of chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Do not leave chemical containers open Use only with adequate ventilation Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal NUNNA CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Handle chemical wastes in a fume hood Minimize contact with and inhalation of chemical waste Wear appropriate personal protective equipment when handling chemicals
188. umn W Output to Waste lt Flow Direction reagents solvents 7 Br From Pump NO Normally Open Port Flow Direction argon w a NI NC Normally Closed Port Fluid Sensor fi Dire F gt IS Com Common Port Check Valve x F C Output to Fraction Collector O Valve 2 way Com NG L T NO The R3 X1 X2 and X3 bottles Valve 3 way have two delivery lines per bottle position Figure 3 1 Procise Valve Diagram 3 4 Valves Functions Cycles and Methods Bottle Vent Valves Three Way Valves The bottle vent valves control argon flow for bottle pressurization and flushing There are 12 vent valves in the Procise Protein Sequencer one for each chemical bottle During chemical delivery these valves remain closed During venting flushing or backflushing these valves are open to allow argon in the bottle headspace to flow to waste The vent valves are also activated by the pressure control system in order to maintain proper bottle pressurization Three way valves are used exclusively for argon delivery Three of these valves are used in the protein sequencer The three way valves provide two different argon pressures from the same manifold inlet line Three way valves control argon input to valve positions 15 24 and 44 and provide high and low pressure settings Table 3 1 Standard Pressures with Three Way Valves Valve Status Function s Pressure Valve 4
189. unts of acetone are added to the solvent A3 and a linear gradient is implemented the absorbance of solvent A3 and solvent B2 will match therefore most of the baseline rise will be eliminated See Chemical Warnings Table on page 1 7 for warnings about B2 A3 and acetone Use the following procedure to prepare the buffer To flatten the baseline by adding acetone to Solvent A3 Step Action 1 Make 1 acetone H O solution Mix 1 mL of HPLC grade acetone and 99 mL of milli Q H O in a 100 mL clean bottle 2 Add 1 mL of 1 acetone H O to 1 liter of solvent A3 buffer Mix well Some HPLC and or PTH columns may exhibit a negative slope in the baseline from DTT to Glu before flattening out in the latter part of the chromatogram The addition of a small amount of phosphate ion to Solvent A usually reduces or eliminates this problem See Chemical Warnings Table on page 1 7 for warnings about sodium phosphate To reduce a negative baseline by adding phosphate ion to Solvent A Step Action 1 Prepare a 1 0 M stock solution of NaH PO or KH PO monobasic sodium or potassium phosphate sodium or potassium dihydrogen phosphate 2 Add 100 uL of the phosphate solution to 1 L of Solvent A to provide a final concentration of 0 1 mM phosphate Note This will flatten the baseline over several cycles and prevent reappearance of the slope PTH Separation 7 7 Optimizing the PTH AA Separatio
190. upling reaction After each PITC delivery there is a short argon delivery to evaporate the heptane Residual heptane would interfere with the reaction of PITC and the sample by keeping most of the PITC in the organic phase The drying time should be at least 20 seconds to insure adequate removal of the heptane It is possible to use two PITC deliveries for coupling rather than three If two PITC deliveries are used the base deliveries should be increased to 270 300 seconds each It is also possible to use more than three PITC deliveries if necessary Examples include a very large amount of sample being sequenced or multiple pieces of PVDF where contact of reagent and membrane may be of concern A base delivery should always precede the first PITC delivery to the cartridge As a first step of coupling in all chemistry cycles R2B vapor is delivered to the cartridge to raise the pH of the sample and deprotonate the free amino groups for reaction with PITC The length of this delivery should be at least 20 30 seconds but can be increased to as much as 120 seconds without negative impact The length of the base deliveries after the PITC delivery can be adjusted but should be at least 120 seconds Avoid making the total base delivery time longer than 700 seconds to minimize the modification of aspartic and glutamic acid residues Under the basic conditions necessary for coupling aspartic and glutamic acid residues are slowly modified by reaction of the
191. ved Del R3 Cart top Del R3 Cart bottom Del R3 Cart sensor Del R3 Waste Load R3 Cart sm loop Load R3 Cart Ig loop Vent R3 Flush R3 Backflush R3 Transfer R3 Cart gas Del R8g Cart top Del R8g Cart bottom Not Available Del R3g Waste Not Available Not Available Vent R3g Valves 6 7 17 18 19 20 34 35 36 37 40 6 11 10 17 18 19 20 34 35 36 37 16 6 7 17 18 19 20 34 35 36 37 40 6 1 6 7 22 6 7 21 55 55 6 11 55 15 3 7 17 18 19 20 34 35 36 37 40 3 11 10 17 18 19 20 34 35 36 37 16 3 1 58 58 3 11 58 15 8 23 17 18 19 20 34 35 36 37 40 8 10 17 18 19 20 34 35 36 37 16 8 23 17 18 19 20 34 35 36 37 40 8 23 16 8 23 22 8 23 21 53 53 8 53 15 7 11 15 17 18 19 20 34 35 36 37 40 9 23 17 18 19 20 34 35 36 37 40 9 10 17 18 19 20 34 35 36 37 16 9 23 16 53 Global Value o O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Funetion List The table below lists the assigned number reagent and the valves involved in a Sensor Function N Z Zz Zz z Z Z Z Z Z Z Z lt lt Z lt Zz z Zz Zz z z Zz Zz Z Z Z Z Z Z Z Z lt lt Z lt Z Global Time Y Z Z Z Z Z lt lt Z Z lt Z lt lt Z lt lt lt Z Z lt Z Z Z Z Z lt lt Z Z lt Z lt lt Z lt lt Number 038 039 040 041 042 043 044 045 046 04
192. wing topics Topic See Page Cartridge Cycle List B 2 Flask Cycle List B 25 Method List B 32 Gradient List B 34 Cycle Method and Gradient Listings B 1 Cartridge Cycle List Cart Begin Total run time 33 20 Step AN OA R 0 PM U GM KM 0 0 0 0 0 NP PN ND PD M MMM N a a aaa 4 so N O dl ER ON O O O MN OO URAN O DAN DOA BR ON O B 2 Cycle Method and Gradient Listings Function 258 146 140 144 106 111 137 136 131 140 26 30 136 140 138 144 143 146 107 137 136 111 64 135 137 140 136 138 131 63 61 148 131 53 51 148 51 Function Name Begin Wash Large Loop Cart Flush Large Loop Cart Wash Cart Solvent Block Wash Input Block S2 Wash Input Block S3 Flush Input Block Flush Cart Solvent Block Dry Cart top Flush Large Loop Cart Load R3 Cart Ig loop Transfer R3 Cart gas Flush Cart Solvent Block Flush Large Loop Cart Flush Output Block Wash Cart Solvent Block Wash Cart Reagent Block Wash Large Loop Cart Wash Output Block S2 Flush Input Block Flush Cart Solvent Block Wash Input Block S3 Del S3 Waste Flush Cart Reagent Block Flush Input Block Flush Large Loop Cart Flush Cart Solvent Block Flush Output Block Dry Cart top Del S3 Cart sensor Del S3 Cart top Cartridge Wait Dry Cart top Del S2 Cart sensor Del S2 Cart top Cartridge Wait Del S2 Cart top Time sec 0 10 40 10 10 10 40 40 60 10 50 5

Download Pdf Manuals

Related Search

Related Contents

Gigabyte GZ-PEBX1-S0R/B0R  CTS-6 User`s Guide  ADE - Ground Polys  Prisma series  Ecommerce Website User Manual  American Weigh Scales EDGE-5K  

Copyright © All rights reserved. Failed to retrieve file