Autoflex Basic User Manual
Contents
1. ass Spectrum m mir Mia Floss 2144312 Default Valinez gt mart 42711 Method Parameters 3000 Pick your method to process your data here AY see window to right and apply 1000 HEIRE 1161862 2000 7 2050 _ 2100 72150 2200 2250 o 2300 miz For pres FL Del sik Value Toon XY Print Spectrum flexAnalysis training online number 2222 File Edit MassList Process Calibrate Annotation Method FAST View Tools Window Compass Help ig BEX E gt B OF dhs ass Spectrum S 112009 0_ 2 1 3 1Ref Raw ii 2144 312 3000 PR Printer MEERA Status Ready Type HP LaserJet 2420 PCL Where 10 79 135 35 Comment Direct printing recommended for PostScript and PDF drivers O Use Screenres S Use Printer res Print with 300 2000 Use pull down menu to Print range Copies C All Number of copies h d Pas Jr choose print mode I e Selection yr spectrum or mass list Layout Spectrum only landscape Orientation O Portrait 9 Landscape 1000 771 577 0 intestato i ierit tti 1000 1500 2000 2500 3000 3500 4000 m z Si un2. EE eos a rar TUER 49 pia T 066513 0007737 7 fono AP 4700 ADDO Select Method Barge 700 1000 2000 J000 Detector Gan Change Mass Range Here Reflects 10x 1 Gan Erhonced Highest For Help press F1 stan 1252 71 1385 55 IF mi 2055 25 ESTE 1550 28 ipia 000000 2 __ 4300 tS Low Piae die Click on Calibration Ta epee High Fl 4000 5000 5000 8000 0 000 10900 Da ih Pang Dargie S 050 1 00 60 x enge 200 GS s Restine Smoot 7 10 Saw 09009 Delay 61352 pts Reltector x 0 00 0 00 Calibration Continued Eg ontrol autoflex RP 4700 mix conditions 700 4000Da par Vew Compass Hep SB RAA RAL M ao e Intent 157059 304 47 8000 A RO Freq 2000 Putin TRAITE RENTRER oose Correct ne ess 5 1700 EELE pe em Ist here 2 n Bm e e lia 5 sesel mo
3. mag 727 488 386 00 771 577 334 00 2143 321 3145 00 2144 312 3621 00 2145 318 2587 00 2146 310 1320 00 2147 344 578 00 2152 304 258 00 2153 323 228 00 3 2165 315 434 00 2166 293 500 00 For Help press F1 Default values gt Zoom AMH flexControl autoflex D Methods Processi Autoflex Basic User flexAnalysis training dy 12 54 PM When Finished View Compass i ABA Kv 9 o th 1 3 8 7 11 13 18 17 1 33 gt m Aud zr xac TERRI EAT DETER Status showing target Cum y dp Vico Zu 4 Instrument Sample Spectrometer Detection Procerum Setup Calwshon Staus Beginning Cut off mass T IET 1 kw M eres Cuppers lan Sacer 2 i Mode Lena 0 00 2 0t peress up Deflection 500 om Fei hi x Calix ation gt gt gt Invaldule 773 Click once to take out target High voltage is switched off by user Reflector AMH x 2143 33 2914 72 PREPARING Place target on Maldi prep bench with the plastic cover and click the target in butt
4. on target Mix 1 of analyte solution with 5 uL matrix Deposit 1 uL on target control sample Mix 1 uL of calibrant matrix mixture with remaining analyte matrix mixture Deposit 1 uL on target Record mass spectrum and recalibrate the mass scale as described previously Multiple tries may be required to find a useful calibrant analyte ratio Common Protein Calibrants M H mono 190 05043 2 mono 379 09303 heme mono 616 198 desArgBrady mono 904 468 Angio mono 1046 542 Angiotensin 1296 6853 GluiFibrinopeptide mono 1570 6768 ACTH 1 17 mono 2093 086 ACTH 18 39 M H mono 2465 199 ACTH 18 39 avg 2466 68 ubiquitin M 3H 3 2855 923 Ins M 2H 24 avg 2867 782 Somat 28 M H _ mono 3147 4714 Somat 28 M H 1 3148 472 Somat 28 M 4 H avg 3149 61 ACTH 7 38 mono 3657 929 ACTH 7 38 avg 3660 12 c equ M 3H 3 Ubiquitin M 2H 2 4 avg Ins bov M H avg Cyt c equ 2 2 Lysozym_ M 2H 2 Myo_equ_ M 2H 2 Ubiquitin M H _avg trypsinogen_ M 2H 2 Cyt_c_equ_ M H Lysozym_ M H CarbAnhyd_IlM2H Myo_equ_ M H trypsinogen_ M H CarbAnhyd equ 2M H BSA_ M 2H 2 Trypsinogen 2M H BSA_ M H 4121 03 4283 446 5734 557 6181 048 7153 60 8476 780 8565 885 11991 47 12361 088 14306 20 14513 37 16952 551 23981 93 29025 74 33904 09 33216 58049 58 66431 A Word About Is
5. B E F G H Indicator e arget Loading Button IL M N Laser is preparing CES spot 420 emen not ready when ready it Select Spot 4 T 0209519 0003797 7 J et flexControl protein 2k 9k mar052008 p Select Method m AutoXecute Sample Carrier Spectrometer Detection Processing Setup Calibration Status Mass Range Selector Instrument specific settings Low Range Digitizer Laser Attenuator Laser Focus Medium Range Dffset 76 Offset 0 2 O High Range Range 20 Range 1001 Trigger Level 100 B vae 40x h O O S e r Digital Linear cnt C Laser Digital Off Reflector Electronic Gain Button Definitions cnt Frequency 200 0 Hz x OffsetLin OffsetRef r a t r F r CANA regular 100 mv 100 200 mV full scale le e e le e Linear 400 00 enh 50 51 100 mV full scale Automation Reflector 1400 00 highest 25 25 mv 50 mV full scale Autoteachin Laser standby Linear AMH PREPARING SIN E FlexControl autoflex fm D Methods Processi Bruker Daltonics Wi m FlexAnalysis Microsoft PowerPoint Suggested flexControl Methods Open flexControl Method FAST psm 1 FAST CID pasm Mu Recent LN ProtMix par Documents LP amp
6. acceptor donor site Cannot photoionize with N laser General Sample Guidelines Purify analyte if possible Analyte should be 5 100 in concentration ZipTips can purify dirty samples and are in MSF Use only volatile solvents buffers MeOH acetone CH4CN THF Cc Hg formic acid etc strength lt 30 mM e g 0 196 v v Acidic conditions required for proper crystallization of many matrices Lack of acidic conditions can be overcome in some cases Need at least 2 uL Instrument Diagram Target E 355 nm Nd YAG laser Reflectron Linear Detector Extraction Reflector Plate Flight Detector Tube Entrance Linear Mode Target E 355 nm Nd YAG laser Reflectron Linear Lens Detector Linear mode is used large 23 5 Extraction Reflector kDa molecules or exceedingly Plate fragile species oligosaccharides It Tube is capable of 4 000 resolving power Entrance 3 2 kDa 1000 RP 9 12 kDa Reflectron Mode Target v 355 nm Nd YAG laser Reflectron Linear Lens Detector Extraction Reflector Reflectron mode is used for small Plate Flight Petector species lt 3 5 kDa and is capable Tube of 11 000 resolving power 3 2 Entrance kDa MALDI Advantages Technique is relatively simple Volatilize and ionize labile molecules Imagine electron ioniz
7. amp kDa par E par al RI par PepMix par RP Proteomics HPC par RP Pratflix par Documents PE Computer a File name Network Files of type flexControl Methods par Ift 129 Use the 4 highlighted methods from above zomin Acquire Data on peaks Clicking max cursor to left and Peak info You can get your Dile Ves Took Pi BE oO we B Aus 3 27 TX Ires arh AL 2143 33 Peak Characteristics Peak Ininimation au 21433277 N b Peak with 1 r 55346203 S N 154 1678 of r 1500 Quality Factor 1237165116 1000 500 n 0200512 0002797 7 ze 2140 2150 2160 2170 2100 mz condones CUD AUUU gaed SS Sample Spectrometer Detection Procersing Setup Calbestion Control List 4700 mix assign assigned Smart Ref Cur Messa 1 Thrshore Statist
8. 0 16767720 197000771 ACTH rx 1 17 M 1 Tolerance w ACTH 465 10000 5 From Mass Contre List ACTH aT 1 Meighbor 50 p u t t h e d ate h e re SO It S Lice Des 5nn ppm Calibration Fit Order easy to find your data Number of calibrants 6 39 dew ppm Before Last standi Reflector 214 17 2914 77 Looking at Data in flexAnalysis A flexAnalyeis trainiog online numbor m fem Smooth Spectrum Zoom in x range pdt Process M FAST 4 wwe Cover Help uid SRF PHY ka cab FF 44 uta media AR Print here Delete Mass Zoom in x y range 3000 List Click on Find Mass List to label peaks 2000 1000 4000 150 2000 2 3000 400 mir 7215 Xu 21423 21500 2144 3 32100 21453 ey 00 14k 3 13400 7141 3 EI 183 3 iB 5 3 4 V OO MEO Eat Processing Data flexAnalysis Fe training mE i 18 de Massiut Process Annotation Merhod FAST Took Window Compair rH 305 4 ad ncs
9. 2500 3000 3500 nominal mass Da Dependence of the fractional masses of predicted Caulobacter tryptic peptides on their nominal masses The red squares represent the locations of the matrix alkali cluster masses Figure from Karty et al in J Chrom B v782 pp363 383 2002 Sample Prep Tricks Ziptip to clean up dirty samples for peptides lt 3 kDa C for peptides proteins 3kDa Elute directly into matrix for added sensitivity ZipTip instructions on MSF website f CCA liquid turns yellow pH is too high Spots from non acidic CCA do not crystallize correctly Add a little 196 v v or 1096 v v TFA to lower pH f sample needs base for solubility try over layer method Dissolve sample in or other volatile base Place 1 uL of sample on target let dry completely Deposit 1 uL matrix over top of dried sample Sample Prep Tricks 2 Non aqueous over layer Make 1 uL spot of matrix on plate let dry Deposit small amount of sample in volatile solvent e g CHCI acetone You can even do internal calibration this way Put calibrants in matrix spot For better mass accuracy let voltages stabilize 5 10 minutes before recording data Sample Prep Continued Samples for MALDI TOF analysis need to meet certain requirements for obtaining good spectra The more careful you prepare samples including early steps of isolation and preparation the more likely a succ
10. Autoflex Basic User Manual Last Updated August 26 2009 What is the Bruker Autoflex Time of flight mass spectrometer lons of given same kinetic energy heavy ions travel slower than lighter ones Two modes of operation Linear for labile molecules or anything bigger than 5kDa Reflectron great for small molecules and peptides Capable of limited MS MS Instruments in Proteomics R amp D Facility are MUCH better for MS MS MS MALDI LDI source 384 position target plate 1 spot size 355 nm frequency tripled Nd YAG SmartBeam laser Can photolyze labile groups e g FITC labels Can analyze positive or negative ions Same spot What Samples Can It Run Biopolymers Peptides proteins DNA RNA oligosaccharides Organometallic complexes Organometallic salts work great Some synthetic polymers Polypropylene glycol PAMAM dendrimers Polycyclic aromatic hydrocarbons with Molecules that photoionize upon irradiation by 355 nm laser Porphyrins Organometallic complexes What Samples Can t It Run Dirty samples Significant concentration of involatiles Glycerol urea most buffers many detergents Alkali metal salts can be quite problematic RNA DNA analyses require extensive desalting Molecules with significant vapor pressures Instrument is held at 10 torr Molecules that do not ionize in source Lack charge
11. Std dev ppm Before Last Fit 1 4 fe Last Fit F Help poses F1 Xx m APIS hh 057 start Li Online Billing Always enter the samples online first before you run your sample s so you can put your data base number in with your sample name Goto http msf chem indiana edu default htm to enter your sample s Log samples you run on the paper sheet next to the printer
12. ation on a protein MALDI creates very simple mass spectra lons are usually or Only 1 3 charge states are observed Usually 1 charge state for peptides 4 kDa MALDI ideal for time of flight analyzers Theoretically unlimited mass range 130 kDa done here MALDI is very rapid 1 min spot Low sample consumption 1 uL Wide array of matrices available for different analytes Some Common MALDI Matrices a cyano 4 hydroxycinnamic acid Molecular Formula farulic acid sinapinic acid SA EX PEE COLO C N C G 2 5 dihydroxybenozoic acid DHB 3 hydroxypicolinic acid HPA tetracyanoquinodimethane TCNQ Molecular Formula 2C H O Molecular Formula C H NO Molecular Formula C H N CCA Mlatrix Good matrix for compounds 10 kDa Makes relatively homogenous flat spots Mix 10 g L in 45 CH4CN 596 CH3 CO 0 1 instructions on balance Combine 1 part analyte with 5 parts matrix Ratio can be adjusted depending on sample Deposit 1 uL on target and let air dry FA and SA Matrices Matrices for compounds 10 kDA Spots are not homogenous or flat Crystallization often must be assisted tap spot Sigma premix SA directions on balance spots are made by mixing 5 1 matrix analyte Deposit 1 on target no tapping required Mix 0 15 M matrix in EtOH alternate method 32 4 g L
13. check mark to the left Calibrant List 4700 mix masses from 904 3660 Dextran 680 2350 Protein Mix F masses from 2465 8465 MSF Big proteins masses 6181 132861 Porphyrins 4700 neg 902 3658 negative ion You can make your own lists see Jon or Angie Saving Data Data Path should always be Data Group User N thexcontrol auigtlex RP 4700 mix condiiems 700 400000 par i CI ook He Click the PREMO ve 8 asa 42s A22 e 2 ao ee your sample name here Save As button to bring up the Save pectru m To 5 TP File Window Ege Save File ill tomatically Comment Lire online we Fiore Pah DADs Mart Spec Labor Karl farle Haa 1 Uorzzrxct roGmmaonomr IFTE Come ONES 7 2130 2180 2150 zB 2170 2180 mr ResConirot HP 4700 mex 7094500 Autdsecube Sample Canes Spectromete Detection Proceming Statur Celestion Sraa Control LHE 0 4700 mix Eds Automatic interactie Cur Massie Emits Peplide gerang bradykinin DT EN ETT 7 w Angeenun M ISR 1 000072 Zoom Range all w urf brnepeptide 00
14. essful analysis will be Here are some guidelines of which kind of treatment is advantageous for mass spectrometric analysis and which is not 1 Avoid the use non volatile agents like salts NaCl CaCl2 2 4 detergents Tween Triton SDS chaotropic agents Urea Guanidinium salts and non volatile solvents like DMSO DMF or Glycerol 2 If you can t avoid these agents purify Dialysis ZipTips and RP HPLC are good purification methods if you use volatile solvents and buffers e g 0 196 v v trifluoracetic acid 10 mM NH4HCO3 After purification lyophilize if possible lon exchange beads may work well for salt removal 3 Suitable solvents are ones that are volatile For sample work up and purification water ammonium hydrocarbonate ammonium acetate ammonium formate acetonitrile trifluoroacetic acid Sample Prep 3 4 Quantitate the sample you are going to provide for analysis by methods like photometry e g OD Bradford assay and ELISA HPLC is useful since it allows for purification and quantitation in a single procedure The range for many samples preparations is not very large therefore it is necessary to have a good estimate of the sample amount because the sample amount may need to be varied on the target 5 The total amount of sample needed for MALDI analysis depends on the sample type For small mw peptides 1 000 or less the minimum amount needed for analysis is 16 picomoles microliter The minimum for
15. for FA 33 6 g L for SA Mix 7 parts analyte 3 parts matrix put 1 uL onto target Wait 30 sec Tap spot with pipette tip until tiny crystals form Stir crystals around so entire spot is covered with crystals SA and FA spots require more laser power than CCA HPA Matrix Used for oligonucleotides 7 mg in 50 uL SCX NH4 resin suspension 50 uL acetonitrile SCX resin desalts matrix and sample Deposit 1 uL resin suspension onto target Let resin dry Add 1 uL each of analyte and matrix solution to dried resin spot Dry sample using heat gun DHB Matrix for Saccharides Mix up saturated DHB in ethanol Mix 1 1 or 1 4 matrix analyte Deposit 1 uL of mixture onto target Tap spot for good crystallization see FA SA page DHB spots require much more light than CCA TCNQ matrix for PAHs Sample prepared without solvent Combine 50 500 parts to 1 part sample Place mixture and 3 steel BBs into a PCR tube Cap tube and tape to vortexer Vortex for at least 5 minutes Apply small bit of powder to target with the back of a wood Q tip requires more light than CCA lons made by charge exchange not protonation Mass Axis Calibration TOF a m z is not practical m z A TOF B TOF C Constants are determined by recording mass spectrum of known compounds A variety of calibration mixtures are available Instrument can be externally calibrated for quick experimen
16. ical Peptide bradykinin 4 46800 90446608 2 4000192 w Angetensn 000172 Zoom w Gl 1570 89720 LSP gung w nipi L74M 1 Peak Assignment Tolerance w 2M 5l0830 2465 15858 4 Prom Mass Control List v ACTH 38 MATIN SLEE 1 Neighbor 50 ppm Defined ppm Caltestion Order Linear 8 Quadrate Harmiber oi ani Sid dev ipank Lack Fil 32 inact Fil 4 Properties Fer Help press Reflector x 24173 0913 7 E me Anas Leer How 4700 rix condithoms 700 4000Da par Dipl ew Compass Heb to Calibrate Pid 8 AAS 4 49 m 2 an g Add JA 0 olete 2 202 lt 0 eT yy D eNe tee Eee eT eee ee 22 2 2 20 2 ES 2 22 2 2 2 E 1 eos 2 2 mus 200 nasi ww eo 2
17. mw 20 000 or less is 60 picomoles microliter For 66 000 mw the minimum amount needed for analysis is 160 picomoles microliter Therefore the larger the molecular weight the more sample is needed 6 Give information like structure sequence molecular weight type of compound biological activity chemical reactivity pH sample amount concentration describe purification isolation with focus on relative agents solvents known or suspected impurities suitable solvents hazardous properties radioactivity carcinogenicity poison or explosive nm m Maldi on Intact Gel Bands 100ul 50uM NH4HCO3 Add 100 ul Acetonitrile Shake for 5 min on vortexer Discard liquid and repeat 3 more times Add 200 ul HPLC water and let sit 10 min Discard water and and repeat Discard water add 15 ul matrix crush gel with tip and let 5 min Spot like normal next to standards and run maldi May not always work try tryptic digest as an alternative 7 My Computer Explorer Za Ll iex Starting the Software Flex Control Screen EE flexControl autoflex protein_mix_F_2k 9k_mar052009 par File Display View Tools Compass Help ww 04 aio Sample Spot View DHB Start Laser Laser Attenuation E Start Shots 0 200 Added 2000 7 Mass Spectrum Window Ei
18. nls Click on Automatic Assign los AEA 19 pe v LESEN Aou Ts 47 1252 B3 3 J gn 75 1750 B4 2093 08 575 431048 G5 1288 67 1550 75 T INIT T ly ilr t il pia s 66 n ee 0209519 7 gt 1000 1520 i 25m 3000 3500 ano mir onto 42001 inox an A Select Method 1 Aubz ecue Sane Detecban Massi conbrol List 4700 mix gt Er Automatic assign Assigned iit Cur Errf Peptide des hrg bradykirin Angoea M 1290 UNI 4 40 0072 bmk E F JOAO 15068771 7 001081 w ACTH 209300620 L 4 Pee Tolerance w MIO 2465 dna ars eae v ACTHU 38 3E57 03308 1 Heighbor S0 pem Calibration ha iiic MEM Click Apply linear Cie tic been a lie Number nf Shide pp Before Last Fit 32 4 For Help press Fl start 1 Fs az reip peaks that are found shou OO Brokar Dakik Peso Pd Id have a
19. on once more to close the Autoflex door Load the RP 4700 mix conditions 700 4000 so the instrument HV comes on and stabilizes for the next user Note Never save a change to a method that s been modified perpe papiy Took Compae Bep wv BABB TW NOT SAY YES 573511 The current method has been modified Do ou want Do sene E Ce A E a H 4 L o Pi IFTE Dames DEI ZA Ear HI ED ketonto poten mix 2 othe Autdsecube Sample Caes Spectromete Detection Procering Spur STER Conil Protein Assign Clear Assigned ret Ref Hanba Cur Miss D 0 ACTH 18 39 MH Ze IUE jaa aert 68 ACTH 18 39 2455 58000 Psi 25 0 TPG 2057 AHT HH MEER ACTHT MMH aw 2600 LJ From Mass Control List ace suh Heighbor 50 ppm ST 573961000 STR 126 1 01208 1 Delil 1000 pem se Ubegun 3 0240517 Calibration Fk Order linear 9 Number of calla 4
20. otopes Instrument can resolve the isotopic pattern of compounds 5 kDa in reflectron mode Many molecular weight calculators compute the isotopically averaged mass Assume 1 1 of C is 9C not C Monoisotopic masses are what we label in reflectron mode data All 12 14N 325 160 Monoisotopic masses NOT usually observed in linear mode for species 2 5 kDa Be aware of which mass your computer program predicted Monoisotopic vs Average Mass Linear Linear Ci 1570 70 3660 18 E E 13C 2 13 iac Reflectron Reflectron uc 570 81 13c 3697 96 s 1560 1570 1580 3650 3660 3670 m z Th m z Th Interpreting Data Mass Defect Atomic weights are not integers except 2 14 0031 Da B 11 0093 Da 1 0078 160 15 9949 Da 18 9984 Da gt 55 9349 Da Difference from integer mass is called mass defect or fractional mass Related to nuclear binding energy Sum of the mass defects depends on composition H N increase mass defect Hydrogen rich molecules have high mass defects e Eicosane C H 282 3286 F Na decrease it Hydrogen deficient species have low mass defects e Morphine 285 1365 Average peptide n C 4 H 3601 4850 04 Peptide Mass Defect Figure 1 0 0 9 0 8 0 7 0 6 055 0 4 0 3 fractional mass 0 2 0 1 0 0 1000 1500 2000
21. ts Internal calibration for better mass accuracy can be tricky to perform Calibration Part 2 The Autoflex is prone to calibration drift Up to 0 25 amu between successive spectra in reflectron mode Up to 2 amu between successive spectra in linear mode Instrument should be started at the beginning of each set of experiments external calibration Calibrant masses need to as precisely defined as possible 3 4 decimal places preferred Calibration can be performed in FlexControl and FlexAnalysis Calibration Mixtures 4700 mix Peptide mixture with masses from 379 3659 Mix F Protein mixture with masses from 2466 8566 Dextran D10 Oligosaccharide mixture with masses from 600 2500 Trypsinogen Myoglobin Protein mixture with masses from 8 476 to 23 981 Other compounds and mixtures are available just ask Jon and Angie External Calibration Calibrants and anlayte are in different MALDI spots Vials of 4700 mix and Protein mix F are in the red MALDI rack Prepare spots as described previously For other calibrant mixes talk to MSF staff Make new calibration spots daily Calibration tips Use 4 calibration peaks and a quadratic fit When using mix F find using only 1 ions gives better results Internal Calibration Spot Preparation Mix 1 of calibrant mixture with 5 matrix This 15 for mix F and 4700 mix other mixtures use different ratios Deposit 1
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