Instrument Handbook - GE Healthcare Life Sciences
Contents
1. 42 Malimenanca tr dana 31 P EE A rr reload bis 24 47 POWER Idle ri 23 38 precautions aia ib is 17 preventive maintenance ioa hela sk datus Put as e 32 DFInelples DSSlC arcada dear 12 PSU PE M m ET 45 Q quadratic reflectron voltage ooooncccnncnconncnacinnnrnnnconcncananconoss 44 R O 11 esee EN RO ETT UNUS 14 44 regulatory requirements agere deese Fa cebat c dat or Cr viens 4 replacing the mains TUSSS 2s bete bor odo ec pret il 34 S safety ADES ira c 18 DIC CAMO metidos alan MER ec a 17 WANES a A i dint SO e uta is 19 Scis Mises oci ME 20 Eee 50 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Index I MOS tl rd 17 sample entry lOCK cisdustesetisea zai sdacut d EXE a adus dete 31 40 SamMplesSide seriada 10 24 40 INTO c 24 OKIENNE dd 42 samples introducing into instrument erica rd 24 schedule user maintenance ooo eee eee sse 28 shutdown ONES ilr Dc 26 Spare Dolls soda e uk ded vnda fice urit A Ur ERE ER UTE 39 LUV AAS IS TE 39 OUT MP 29 42 EEE elo sar HH 33 joie EE 4 spectrum tz Mn dla a 45 starine the Instrument usar dori talon daa fete em osi 23 T teennicaldes c HDEHOL sra 43 theory time of flight 13 MA e a cha 44 time of flight ajjiele mee 12 A A ETA 13 A a a RA 45 A a a a E 29 TOUS IN uti a 39 troubleshooting SUI e sra 36 U user mantela C B ias 28 V Vacuum IMaIcator AS 24 38 vacuum system DOCK2. 6 1 Description 6 1 1 The Ettan MALDI ToF instrument The Ettan MALDI ToF is a mass spectrometer that can operate in linear mode or in reflectron mode using a harmonic reflectron that increases resolution and sharpens time focusing Samples are placed on slides and introduced via the sample entry door A sample entry lock ensures that the flight tube vacuum is preserved during sample exchange Air outlet Vacuum System A outlet C qa Flight Air outlet tube N Air outlet Sizes NN xx gt Indicators cc cc Mains inlet panel Sample entry door Air inlet Fig 6 1 The Ettan MALDI ToF instrument 6 1 2 Cooling A number of fans mounted in the instrument provide a flow of cooling air that exits through the vents at the rear and top of the instrument These air inlets or vents must not be blocked do not place papers underneath the instrument Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 37 6 Reference information AE 6 1 3 Vacuum system outlet 38 To protect users and the electronics from any oil mist a vacuum system outlet is situated at the top of the instrument CAUTION The vacuum system outlet should be vented into a fume hood evacuating ambient air 6 1 4 Indicators on the front panel The front panel of the Ettan MALDI ToF contains the following indicators Blue mains power is connected to the instrument and the mains switch is on Vacuum Blue the vacuum has rea
3. manufacturer s manuals Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 47 Index A ACCES SOMES A e a a Active indicator ui dada didas a alt wasser ate ervD tope vacuum systeM zsiuskceuietalutontietun queo sates C calibration peptides ita cleaning procedures ee closing down the system COMMECIONS nadas A A ap futs ad descripto dui ioter lo iaa diagnostic guide esee disposal procedures eee documentation ascender deseo ceci lali E emergency procedures een Ettan MALDI ToF instrument ESCUDO aa Ettan MALDI ToF system F fragment JOD Spectra ra ds fuse PEDIC ING AORTA OP USO M SPECICATION ac H harmonic reflectron cene indicator ACUVO ral aR uoc c eaten A II WCNC ALOIS ds interlockS introducing samples at Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 49 Index 6S SR ee ee CR Te ee oe 9 L cr 18 logging in to the MS software occcocccncccncccncccnccnnnccnccnnniconananess 24 lone tern slip OE rias 26 M A O O re e o O aA 24 4 mains fuse So m 34 FMA CHANCES iia 2 eia gi er 30 cic met Ta ao 30 A E 31 SS Tom t a a idas 32 0 operating instructions basic 23 Operan e DINICIDICS an tados 43 Ordering AMIONMIALION A 42 org H
4. applications The Ettan MALDI ToF MS Software Online Help contains detailed descriptions of all software menus and dialogues The Ettan MALDI ToF manuals are delivered as spiral bound books or binders as indicated in the margin Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 15 Safety instructions E 2 Safety instructions IMPORTANT To avoid any risk of injury the instrument should only be operated by properly trained personnel and always in accordance with the instructions provided The purpose of this chapter is to describe safety precautions and present all safety labels that are attached to the instrument Built in safety functions are described and emergency and disposal procedures are included 2 1 Safety precautions 1 Read this entire manual before using the Ettan MALDI ToF instrument 2 This instrument is designed for indoor use only 3 The instrument must always be used with the protective earth lead of the power cord correctly grounded to earth at the mains outlet 4 To permit sufficient cooling ensure that the vents at the top rear and bottom of the instrument are not covered 5 Do not operate the instrument in extreme humidity above 95 Avoid condensation by letting the unit equilibrate to ambient temperature 6 Keep the instrument dry and clean Wipe regularly with a soft damp tissue Let the instrument dry completely before use see Chapter 4 7 Any equipment connected to th
5. fuse 230 V T 4 A 2bO V Mains fuse 115 V T 8 A 125 V Table 4 4 Spare parts replaceable by the user Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 33 ED Maintenance 4 10 1 Replacing the mains fuses WARNING Always disconnect the mains supply before removing the mains fuses For continued protection against risk of fire replace only with fuses of the same type and rating Replace the mains fuse as follows 1 Disconnect the mains power cable from the Ettan MALDI ToF 2 Insert a small screwdriver in the slot at the fuse cover 3 Pull out the fuse holder 4 Replace the fuse 5 Insert the fuse holder Note that the arrow beside the selected voltage range must point at the white line on the mains connector Fuse holder position for 230 V Replace AAOZL OLL AO0S2 30 S3A19gISNJ SJA Env Er Ere a RENO INS SASNA A0S HLIM AINO ETE 220 240V Y nd s3a Fig 4 2 Replacing the mains fuse 34 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Troubleshooting qeu 9 Troubleshooting This chapter provides basic diagnostic and troubleshooting guides The diagnostic guide is based on the error messages that are displayed by the Ettan MALDI Control software The basic troubleshooting guide focuses on error symptoms related to instrument operation For errors related to the appearance of mass spectra e f you have a problem with sample preparation refer to the Ettan MALDI ToF Metho
6. log in dialogue 2 5 3 Restart Procedure In the event of system shutdown due to power failure emergency stop or process interruption any malfunctions must be rectified before the Ettan MALDI ToF is restarted To restart the Ettan MALDI ToF follow the Start up instructions provided in Chapter 3 2 6 Disposal procedures The sample slides should be disposed of as metal waste To ensure that your samples are not contaminated always use new sample slides E Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 21 ID Safety instructions 22 2 7 Recycling The Ettan MALDI ToF contains the following materials Stainless steel Aluminium Ceramic Plastic Acetal POM Polyetheretherketone PEEK Fluoric rubber Viton Electronics components Vacuum oil When taking the Ettan MALDI ToF out of service the different materials must be separated and recycled according to local regulations Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Basic operation E 3 Basic operation This chapter provides basic operating instructions for the Ettan MALDI ToF instrument including start up acquisition introducing samples and shutdown Refer to the Ettan MALDI ToF User Manual for instructions on performing analyses The start up instructions provided in this chapter assume that the instrument and the PC have been correctly installed by Amersham Biosciences personnel When planning analyses bear in mind that the instrume
7. standards This product meets the requirements of the EMC Directive 89 336 EEC through the harmonized standard EN 61 326 1 1997 A1 1998 WARNING This is a Class A product In a domestic environment this product may cause radio interference in which case the user may be required to take adequate measures The CE symbol and corresponding declaration of conformity is valid for the instrument when it is used as a stand alone unit or connected to other CE marked Amersham Biosciences instruments or connected to other products recommended or described in this manual and used in the same state as it was delivered from Amersham Biosciences except for alterations described in this manual All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request Ettan is a trademark of Amersham Biosciences Limited Amersham and Amersham Biosciences is a trademark of Amersham plc Microsoft and Windows NT are trademarks of Microsoft Corp Amersham Biosciences AB 2000 All rights reserved Office Addresses Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences Inc 800 Centennial Avenue P O Box 1327 Piscataway N J 08855 1327 USA A
8. 18 1144 00 Edition AB 41 6 Reference information 64 Ordering information To order accessories for Ettan MALDI ToF quote the designation and Code no when contacting your Amersham Biosciences representative Sample Slide Kit 18 1147 18 Matrix chemicals amp cyano 4 hydroxycinnaminic 17 6002 80 acid 4 HCCA 2 5 g 2 5 dihydroxybenzoic acid 17 6002 81 gentisic acid or DHB 2 5 g Sinapinic acid sinapic acid 17 6002 82 trans 3 5 dimethoxy 4 hydroxycinnamic acid 2 5 g Calibration peptides ile Angiotensin III acetate 1 6002 83 salt 1mg hACTH fragment 18 39 17 6002 84 adrenocorticotropic hormone 0 5 mg hACTH fragment 7 38 17 6002 85 adrenocorticotropic hormone 0 5 mg Calibration protein Albumin bovine 10 g 17 6002 86 O ring 42 3 x 3 Viton 18 1150 37 Table 6 4 Ordering information 42 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Reference information 6 65 Technical description 6 5 1 Operating principles The basic theory behind the operation of the Ettan MALDI ToF is briefly described in Sections 1 4 1 to 1 4 3 A laser pulse ionizes the sample and desorbs the ions into the vacuum The high voltage accelerates the ions which then enter the field free drift region flight tube travelling at constant velocity until they hit the detector or in reflectron mode until they enter the reflectron In reflectron mode the ions are retarded final
9. Fig 6 8 Fig 6 9 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB The Ettan MALDI TOF system eer 10 PY DIC aInSeN le SIS TTE 10 Location and layout of rating labels ooccooonccconcconcconcnnononnonons 11 Example of a Mass sDeclr tmu o entente tii 12 The principle of time of flight mass spectrometry 12 Harmonic reflectron principle The reflectron voltage has a quadratc cnaracteristie ada d oet echtes 14 Pinch hazard warning at sample door ssssesssesse 18 safety label on the connector panel eese 18 Checking the air OW sac sari csanc mat iesus aao cet edet nds 30 Replacing the mains WS esr EO S T o T 34 The Ettan MALDI TOF instrument eene 97 Connector panel with mains switch eeeeeeee 39 The sample entry lock eese 40 Etenediwell sample SIM oras roep natia clin Naa 40 Basic operating principle of the Ettan MALDI TOoF 43 Principle of the quadratic field reflectron 44 Time separating ions created by post source decay of a recurs TOES asper ovp iE rA E burried OS BR I URDU REI ED PAPA RN dE PASSE RAE 45 Block diagram of the Ettan MALDI ToF instrument 46 ie Vacuum SIS ranita 47 vil Introduction eu 1 Introduction MALDI TOF matrix assisted laser desorption ionization time of fli
10. Handbook 18 1144 00 Edition AB Maintenance EN 4 Maintenance 4 1 Introduction Regular maintenance is important for safe and trouble free operation of the Ettan MALDI ToF The user should perform daily and monthly maintenance Preventive maintenance should be performed on a yearly basis by qualified service personnel This chapter provides instructions for user maintenance a schedule for preventive maintenance and instructions for replacement of the mains fuse Contact your Amersham Biosciences representative for more service information WARNING HIGH VOLTAGE Always exercise extreme caution when handling this instrument The Ettan MALDI ToF contains a power supply producing lethal high voltage of more than 20 000 V DC inside the instrument Always shut down the instrument and disconnect the mains power cord before performing any maintenance operations WARNING NO SERVICEABLE PARTS INSIDE Do not open covers Service and maintenance should be performed by qualified personnel WARNING Do not touch or inhale the thermal breakdown products of fluorinated materials which may be present if the rotary pump has been heated to 260 C or more The pump may be overheated in cases of misuse or malfunction Fluorinated materials can be present in oils greases and seals Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 27 ED Maintenance 4 2 User maintenance schedule Table 4 1 lists the maintenance operations that sh
11. Qa proteomics Ettan MALDI lor Instrument Handbook Amersham um 18 1144 00 Y Biosciences Important user information All users must read this entire manual to fully understand the safe use of Ettan MALDI ToF WARNING The Warning sign highlights an instruction that must be strictly followed in order to avoid personal injury Be sure not to proceed until the instructions are clearly understood and all stated conditions are met WARNING High Voltage A The High Voltage sign highlights an instruction that must be strictly followed in order to avoid contact with lethal high voltage above 1 kV Be sure not to proceed until the instructions are clearly understood and all stated conditions are met WARNING Laser Radiation A The laser radiation sign highlights an instruction that must be strictly followed in order to avoid exposure to hazardous laser radiation Be sure not to proceed until the instructions are clearly understood and all stated conditions are met Caution The Caution sign is used to call attention to instructions or conditions that must be followed to avoid damage to the product or other equipment Be sure not to proceed until the instructions are clearly understood and all stated conditions are met Declaration of conformity Safety standards This product meets the requirements of the Low Voltage Directive 72 23 EEC through the harmonized standard EN 61 010 1 1993 A2 1995 EMC
12. be defined and only this ion and its fragment ions will pass the gate The fragment ions can then be analysed to obtain sequence information Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 45 6 Reference information 46 6 5 3 Block diagram The block diagram in Fig 6 8 shows the main subsystems of the Ettan MALDI ToF instrument A high vacuum is required for proper function of the instrument As indicated in the block diagram the vacuum system evacuates the ion source and the flight tube including the ion gate the reflectron and the detectors An external computer supervises the electronics of the instrument and controls the transfer of data from the Ettan MALDI ToF The instrument hardware controls the sample exchange and the acquisition of mass data Electronics crate Acquisition I O Control Control External Electronics computer Motors sample tray inlet door Evacuated parts Pulsed Reflectron extraction lon gate and unit detectors lon source Target Power Vacuum supplies system Fig 6 8 Block diagram of the Ettan MALDI ToF instrument 6 5 4 Vacuum system The vacuum system operates continuously while mains power is connected to the instrument Refer to the block diagram in Fig 6 9 for the following description The rotary pump forms the first stage of the vacuum system backing the turbo pumps and evacuating the sample entry lock inlet chamber Vacuum valves a
13. ched the operational level below 1 4 x 10 mbar Active Blue the high voltage supply is on an experiment may be running Table 6 1 Indicators on the Ettan MALDI ToF front panel Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Reference information qu u 777 6 1 5 Connectors The connector panel is located at the rear of the instrument The mains power inlet with the mains switch and mains fuse is located on the connector panel see Fig 6 2 Nitrogen inlet L Cc Mains power inlet Prepared for slide loader Connector in e gt panel PC 1 za PC2 PC cable i Fig 6 2 Connector panel with mains switch The communication ports PC 1 and PC 2 are intended for service purposes only The PC used for acquisition of mass data 1s connected via a specially matched cable which is permanently installed in the Ettan MALDI ToF instrument CAUTION Do not under any circumstances connect any equipment to the PC 1 or PC 2 port Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 39 6 Reference information 40 6 1 6 Sample entry lock The sample entry lock enables the vacuum in the flight tube to be maintained during sample exchange When Process Open door is selected in the Ettan MALDI Control software a sample manipulator moves the sample holder from the target area to the sample entry lock Solenoid valves then open and vent the sample entry
14. cinnonenrooronoannenononoronccanonenoncns 29 2 5 CN ANS POC CO UNCS io 30 4 5 1 Cleaning the Instrument oo ce th a deor E RH 30 4 6 Fae NUM ACS traca lodo 30 4 7 Working with the vacuum system eee 30 AF we C IUE Pm S Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Contents vi A S Oie MAME aC aaa tied does ts 31 4 9 Preventive maintenance eesesseeeen m eene de Z TO Replacine spare Pal scr tili 33 A103 Replacing the Mains TUSS ioc ie aaie 34 Troubleshooting SM MN qai Seatac a ee ee ae el Vie aa eee 35 Ie MOUDE ne 36 Reference information A O II 37 Od The Ettan MALDI TOF SUITE ente oh Prin 9 6 1 2 CODD xs MU CE tC AL NM UN UE et 3 7 JS Vacuum yS On OU O a ai Us 38 6 1 4 Indicators On the TONE PI eh 38 OdG Qiolgie 0ii0 s PE 39 6 1 6 SO SNO UM EE 40 6 2 Sample slides ainda ta lis 40 63 vec S DC 4 6 34 Fechnicat SDCCIICAUONS sree crest oto tette ooa Du Feat ted ette 41 6 3 2 Regulatory TEQUES A A D e eet el 41 0A Dre ne IOMA EM NE OO OS 42 Ox dechnicabdeseriptloll atudassdasss umet eiue uM ED UD UL MINE 43 5 5 ET CO NAAA A 43 6 57 Mero OC UOT ERR RR 44 6 5 3 BIOK OOT I aaraa a TA 46 6 5 4 Vac OBSS Vara 46 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB List of figures Fig 1 1 Fig 1 2 Fig 1 3 Fig 1 4 Fig 1 5 Fig 1 6 Fig 2 1 Fig 2 2 Fig 4 1 Fig 4 2 Fig 6 1 Fig 6 2 Fig 6 3 Fig 6 4 Fig 6 5 Fig 6 6 Fig 6 7
15. controlled via the software SADA ns I H A Fig 1 1 The Ettan MALDI ToF system Samples are spotted or positioned onto a sample slide with a number of sample wells Fig 1 2 Typical sample slide Ettan MALDI ToF allows selection of both positive and negative ion operation as standard A detailed description of the Ettan MALDI ToF instrument is provided in Chapter 6 of this manual Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Introduction au E 12 1 Rating labels The rating labels are located on the connector panel at the rear of the Ettan MALDI ToF instrument see Fig 1 3 Pay attention to the component ratings These ratings determine the electrical safety hazards in the equipment connected to the supply voltage There are however other hazards which may be more severe see Chapter 2 Safety instructions Voltage 220 240 V Mains switch WARNING Mains power Vw For continued protection against risk inlet E MEE of fire replace only with fuse of the Bas ange specified type and current ratings 100 120 V Mains tuge Fig 1 3 Location and layout of rating labels 1 3 Accessories The Ettan MALDI ToF instrument requires the use of specific sample slides which can be ordered from Amersham Biosciences In addition MALDI associated chemicals and calibration peptides are available Ordering i
16. cuum pump at Hardware turbo pump controller speed relay Sample inlet door closed Hardware microswitch Cables to door sample motor Hardware loop in connector vacuum pumps connected request Source turbo vacuum pump at Hardware turbo pump controller speed relay Tube cover in position rear panel Hardware microswitch Software high voltage enable Software hardware logic Table 2 1 Interlock conditions for high voltage supplies and laser Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Safety instructions E ES 2 9 Emergency procedures 2 9 1 Emergency shutdown CAUTION Do not switch off mains power unless there is an immediate risk of physical injury The Ettan MALDI ToF may be damaged by a sudden loss of power In a situation where there is a risk of physical injury turn off the mains switch at the connector panel of the Ettan MALDI ToF If the sample inlet door 1s moving it will immediately stop when power is interrupted 2 5 2 Power failure routine In the event of a power failure the experiment is interrupted in an undefined state However the data collected up to the power interruption is saved To protect the flight tube from diffusing of vacuum pump oll a valve is opened and the flight tube is vented to atmosphere or if connected to a nitrogen supply When power returns 1 The Power indicator on the instrument lights 2 The fans and the vacuum pumps start 3 The PC starts and displays the
17. d Handbook e The instrument may need calibrating see the Ettan MALDI ToF User Manual for instructions 5 1 Diagnostics Error message Possible cause Corrective action Moist in sample Incorrect sample preparation Refer to Ettan MALDI ToF Method Handbook Leakage Dust or other object Check for foreign objects at the obstructing the movement of sample inlet door Clean the the sample inlet door O ring If necessary See Section 4 8 Table 5 1 Diagnostic guide Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 35 ED Troubleshooting 52 Troubleshooting Error symptom Possible cause Corrective action Insufficient vacuum or long Leakage at sample inlet door Open sample inlet door If down pumping time after Pirani and magnetron sample exchange pressures stay within specifications check for foreign objects at the sample inlet door Clean the O ring if necessary See Section 4 8 Leakage at vacuum Call APBiotech service connections Oil on the floor below the Leakage at the rotary pump Call APBiotech service instrument Can not start a run Operational vacuum not yet Wait until Vacuum indicator established lights before starting the run Apparent low sensitivity Worn out detector or laser Run standard sample Call APBiotech service if problem persists Table 5 2 Troubleshooting guide 36 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Reference information qu 6 Reference information
18. d dust should be removed WARNING Do not drop any liquid into the air vents at the back and top of the instrument If this has happened shut down the Ettan MALDI ToF Section 3 3 and disconnect the mains power cable Let the instrument dry completely before reconnecting to mains power 1 Wipe the instrument cover with a soft damp tissue 2 If needed use a mild detergent to remove stains 4 6 Fan maintenance Check that cooling air flows through the instrument exiting through the vents at the rear and top of the instrument Air outlet Air outlet Air outlet Air outlet Fig 4 1 Checking the air flow If the fans are noisy service may be required Call your Amersham Biosciences service representative 4 7 Working with the vacuum system CAUTION Cleanliness is important when working with the vacuum components Any contaminants or moisture inside the flight tube may increase down pumping time or reduce the end vacuum Always wear powder free gloves when working with the vacuum system 30 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Maintenance EN 4 7 1 Sample entry lock The sample entry lock enables samples to be exchanged without venting the flight tube However the sample entry lock must be evacuated when the sample door is closed before the sample can be moved to the target position Avoid leaving the sample inlet door open for long periods of time not more than 2 minu
19. dag atq as 47 o cT 46 Mentdlato OB OL ds 38 esae imc den ld dd 30 pug Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 51
20. e instrument should meet the requirements of the EN 61 010 1 or other international safety standards Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 17 2 Safety instructions 77 2 2 Safety labels The following safety labels are attached to the Ettan MALDI ToF instrument to warn the user of potentially hazardous conditions At the sample door Fig 2 1 Pinch hazard warning at sample door WARNING PINCH HAZARD Make sure that nobody is working at the sample entry lock before choosing the Process Close Door command in the Ettan MALDI Control software On the connector panel Fig 2 2 Safety label on the connector panel CAUTION Read this manual before connecting or disconnecting any cables or opening the covers of the Ettan MALDI ToF instrument WARNING NO SERVICEABLE PARTS INSIDE Do not open covers Service and maintenance should be performed by qualified personnel 18 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Safety instructions E AA 2 3 Warnings WARNING HIGH VOLTAGE Always exercise extreme caution when handling this instrument The Ettan MALDI ToF contains a power supply producing lethal high voltage of more than 20 000 V DC inside the instrument Always shut down the instrument see Section 3 3 page 26 and disconnect the mains power cord before performing any maintenance operations WARNING Do not use the Ettan MALDI ToF in any other way than described in thi
21. e intensities of ions of different mass to charge ratios see Fig 1 4 Since the charge state is usually z 1 or z 2 in MALDI experiments it is possible to identify peaks as representing certain masses Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 13 ED Introduction AAA 14 3 Reflectron The Ettan MALDI ToF includes a harmonic reflectron that acts as an ion mirror see Fig 1 6 The reflectron improves resolution by extending the ion flight path In addition ions of equal mass having slightly different energy will be focused in time thus giving more narrow peaks in spectrum also contributing to higher resolution See Section 6 5 2 for more details Detector Reflectron linear mode voltage 72 Detector reflectron mode Timed ion gate Laser y Sample slide Accelerating voltage x Ye ak e Fig 1 6 Harmonic reflectron principle The reflectron voltage has a quadratic characteristic 14 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Introduction eu 1 5 Associated documentation The following documentation is included with the Ettan MALDI ToF The Ettan MALDI ToF Site Preparation Guide describes the requirements that must be fulfilled before the Ettan MALDI ToF instrument can be installed The Ettan MALDI ToF User Manual contains detailed operating instructions The Ettan MALDI ToF Method Handbook provides practical guidelines and examples of specific
22. egion In the reflectron the ion having the higher energy travels further before turning back and both ions arrive to the detector at the same time Reflectron voltage Same mass differing velocity Detector reflectron mode Time focused ions Reflectron Fig 6 6 Principle of the quadratic field reflectron Separating ions having the same velocity In a PSD experiment the ability to separate ions having the same initial velocity is used to analyse fragments from a precursor ion When formed the fragment ions keep the total kinetic energy of the precursor i e they continue at the same velocity towards the entrance to the Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Reference information 6 reflectron In the reflectron they are separated due to their mass differences and a fragment ion spectrum can be obtained Intact precursor ions also give rise to a peak in the spectrum Because of the Z technology the complete PSD spectrum is acquired in a single run Fragment ions same velocity differing mass Detector reflectron mode Precursor ion RS 2 ot PSD Time separated Reflectron fragment ions PSD Spectrum Precursor P d m z Fig 6 7 Time separating ions created by post source decay of a precursor ion Timed ion gate The Ettan MALDI ToF is equipped with a timed ion gate which is used in PSD experiments The m z for a selected peptide ion can
23. esponds to mass The mathema tics behind this conversion are described very briefly in the next section Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Introduction au ss 14 2 Simplified theory The electric field in the ion source accelerates the ions into a field free drift region The kinetic energy of each ion is proportional to the charge of the ion q and the accelerating voltage V The travel time in the drift region depends on the mass of each ion As shown in Fig 1 5 the ions will be separated so that ions with low mass reach the detector first The time of flight principle relies on the following basic equations qV My Where q ion charge V voltage 1 M on mass v velocity se pp Where t time of flight 2 L length of the drift region flight tube In this simplified model the travel time for an ion is proportional to M 3 la 3 Considering that the ion mass and charge are proportional to the atomic mass of the ion and its charge state respectively M 1 sexi m where M ion mass kg 4 m atomic mass Da q ez Where q ion charge C S e constant 1 60 x 10712 C z ion charge state dimensionless we can express the travel time as m t k wherekz constant Z By detecting the number of ions arriving at different times a mass spectrum for the sample can be calculated and displayed The mass spectrum shows a number of peaks representing th
24. ght see Section 1 4 The Ettan MALDI ToF mass spectrometer is a bench top research grade instrument incorporating a harmonic reflectron and high mass detector Ettan MALDI ToF combines the protein knowledge of Amersham Biosciences with that of experts in protein identification ProteoMetrics LLC and mass spectrometry Scientific Analysis Instruments Ltd to offer a MALDI TOF mass spectrometer for fast reliable protein identification The compact and automated instrument features a high resolution timed ion gate and a quadratic field reflectron that can be used in post source decay PSD studies of protein fragments Examples of applications e protein peptide mass determination e determining the indentity of a protein e verifying a protein identity e identification of interaction partners e studies of various biomolecular problems 1 1 The Instrument Handbook This handbook provides safety instructions technical information and basic operating instructions for the Ettan MALDI ToF instrument In addition maintenance schedules and instructions for user maintenance are included Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 9 ED Introduction 10 1 2 The Ettan MALDI ToF system The Ettan MALDI ToF system comprises the Ettan MALDI ToF instrument and the accompanying control and analysis software which runs under Windows NT operating system on a PC After the sample slide is introduced the system is fully
25. lock to atmosphere Finally the sample door opens and the sample slide can be exchanged Fig 6 3 The sample entry lock 6 2 Sample slides Samples are spotted onto sample slides which are inserted into the sample entry lock on the Ettan MALDI ToF One type is currently available the Etched well sample slide Samples are applied onto smooth spots surrounded by an etched trough Excess sample can drain into the trough This slide is recommended for proteins and peptides Fig 6 4 Etched well sample slide Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Reference information qu 7 7 6 3 Specifications 6 3 1 Technical specifications Mains voltage Preset from factory 100 120 V 10 50 60 Hz or 220 240 V 10 50 Hz single phase Installation category 11 Operating temperature 15 27 C Dimensions including stand 153 x 53 x 66 cm H x W x D Length of flight path 1 2 m linear 2 m reflectron Weight 152 kg Laser Pulsed UV 337 nm nitrogen Class 3A Nitrogen supply Max 0 1 bar 10 kPa Table 6 2 Ettan MALDI ToF technical specifications 6 3 2 Regulatory requirements Ettan MALDI ToF meets the standards listed in Table 6 3 Safety IEC 61 010 1 1990 IEC 61 010 1 A1 1992 and IEC 61 010 1 A2 1995 Laser IEC 60 825 1 1993 and IEC 60 825 1 A1 1997 EMC IEC 61 326 1 1997 and IEC 61 326 1 A1 1998 Table 6 3 Regulatory requirements Ettan MALDI ToF Instrument Handbook
26. ly reversed and accelerated again towards the second reflectron detector See separate description in Section 6 5 2 Detector Reflectron linear mode voltage z Detector reflectron mode Timed ion gate Accelerating ey voltage MO Fig 6 5 Basic operating principle of the Ettan MALDI ToF Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 43 6 Reference information Post Source Decay PSD See Ettan MALDI ToF User Manual for more information 44 6 5 2 The reflectron Ettan MALDI ToF contains a unique reflectron based on patented Z technology By applying a quadratically increasing voltage 1t produces a linearly increasing electrical field see Fig 6 6 This type of reflectron is also referred to as harmonic due to the fact that the ions observe one half cycle of harmonic motion The reflectron has three main properties all contributing to a high resolution e The ion flight path is extended compared with a linear ToF e ons of the same m z ratio having different energies arrive at the same time at the detector time focusing e ons of different m z ratios are separated in time even if the have the same velocity when entering the reflectron Time focusing The ability to time focus ions is illustrated in Fig 6 6 Two ions of the same m z have slightly different energy after the initial acceleration and thus separate during the flight in the drift r
27. mersham Biosciences Europe G mbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences KK Sanken Building 3 25 1 Hyakunincho Shinjuku ku Tokyo Japan Contents Introduction 1 1 The Instrument Handbook Vu Die e c tcu 9 1 2 The Ettan MALDI TOF system esas 10 T2 d QC No E ig WS esc sunt cn uaniencecaneaungnedecamete acer seaman 11 1 4 Time of flight mass spectrometry ene 12 L e iama ica jolie 12 1 4 2 DUOC TCO A dT 13 1 4 3 o Mes LT 14 1 5 Associated documentati ON a aid 15 Safety instructions 2 1 Sale DEC aUIONS croacia 17 ARES AN sues Doleo es ud es e Ook i assu 18 2 9 A tdo 19 24 Buillt in safety TUMCTIONS 3 5 ic il 20 24l E UE 4 961 APPO PPP PO T 20 20 Emergency procedures us a 21 Zod Eme gency SADO Wi a ZA 2 0 2 POWER TAN OT OMT e ect aos esum EU EE E E LAE 21 25 3 PROSTATE IOCCO UIC ost einst ou dudit E Eun oen e Ds und 21 210 DISPOSal DOCUS e RARE ELI a eos 21 A ROO CIN aae a HOP 22 Basic operation 3 1 Starting the INstrument npe ten peo as 23 Se IMtroducines Samples sata reins thai dist dei stetur tur 24 3 3 Closing down the System ie 26 Su Closing down the Ettan MALDI Control software 26 OR WOnS emu A e tot cepe tace bend 26 Maintenance o AO coude E M norms Penile E 2 4 2 User maintenance schedule cocooccccoccncoccnoncnnnnnnnncnnoncnnonccnononos 28 AS S Dee Dalee trie a R 29 4 4 Tools and accessories ocococcccnonononea
28. nformation for accessories is provided in Chapter 6 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 11 ED Introduction Intensity 3 N 1 4 Time of flight mass spectrometry 1 4 1 Basic principles A time of flight mass spectrometer separates 1ons of different masses and produces signals that provide timing information for 1ons at each mass The signals are accumulated and by using statistics the amount of ions at each mass can be represented in a diagram the mass spectrum see Fig 1 4 Intensity Medium mass e e 9 T Low mass O High mass Fig 1 4 Example of a mass spectrum In a MALDI TOF instrument a laser pulse desorbs and ionizes the biomolecules incorporated in a matrix on the sample slide The ions are accelerated by high voltage acquiring a velocity that depends on the mass light ions acquire a higher velocity than heavy ions see Fig 1 5 Entering a field free drift region each ion keeps the constant velocity until it hits the detector lons of different mass can thus be separated in time heavy ions take longer than light ions Laser pulse Sample Detector O 4 Y o me et e ee yd z e lon D o source LL V L length of drift region Fig 1 5 The principle of time of flight mass spectrometry In the mass spectrum the accumulated signals are plotted against mass over charge m z which usually corr
29. nt needs at least 24 hours from power on to establish the required vacuum If the flight tube is filled with nitrogen the down pumping time may be somewhat reduced due to the low water content of the nitrogen 3 1 Starting the instrument WARNING High voltage is present within the Ettan MALDI ToF instrument during an experiment CAUTION Do not move the Ettan MALDI ToF with power on as this may damage the vacuum pumps To start the Ettan MALDI ToF system 1 Turn on mains power to the Ettan MALDI ToF with the Mains power switch at the connector panel 2 Check that the Ettan MALDI ToF is operating lt D Power e the Power lamp should light lt Vacuum e the fans should operate producing a cooling air flow that exits at the rear and top of the instrument e Active 3 Switch on mains power to the PC 4 Start Ettan MALDI Control software by selecting Programs MALDI Software Ettan MALDI Control from the Windows Start menu Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 23 ED Basic operation c Vacuum e Active 24 5 Login to the Ettan MALDI Control software The Control instrument option must be checked Configurations Configuration 1 Username Password Cancel 6 When the Ettan MALDI Control software is running check the vacuum levels displayed in the status bar at the lower end of the screen Pirani2 3e 04 mbar Magnetron 4 5e 05 mbar High voltage OFF Doo
30. nt should normally be connected to mains power continuously to preserve operating vacuum When finishing the day s work you only need to close down the Ettan MALDI Control software 3 3 1 Closing down the Ettan MALDI Control software 1 Make sure that your analysis data have been saved 2 Check that no experiment is running the following conditions should be indicated in the status bar e Instrument status Idle e High voltage OFF e Door is closed Instrument status Idle High voltage OFF Door is closed 3 Check that the Active lamp is off 4 Close down Ettan MALDI Control software by selecting Exit from the File menu 3 3 2 Long term shutdown If the Ettan MALDI ToF will not be used for a longer period of time e Close down the instrument e Cover the instrument to protect it from dust e Keep a steady temperature in the room e Do not move the instrument unnecessarily If nitrogen is used check that the nitrogen pressure is within specifications see Section 6 3 before turning off instrument power The nitrogen gas is used to fill the flight tube instead of venting it to air as this may cause adsorption of water to surfaces within the instrument To close down the Ettan MALDI ToF instrument 1 Close down the Ettan MALDI Control software as described in Section 3 3 1 2 Switch off mains power to the PC 3 Switch off mains power at the connector panel on the Ettan MALDI ToF instrument Ettan MALDI ToF Instrument
31. ons Check all internal and external cable connections Mechanics Inspect and lubricate door mechanism Replace the Viton O ring the Viton Replace the Viton O ring ring Check function of the sample mechanism Check the laser focus mechanism Check the mirror and focal lenses Laser Measure laser power and detect signal Adjust laser 00000 Adjust laser 00000 Electronics and power supplies Measure pulser signals and high voltages Turbo molecular pumps Check bearings Rotary vacuum pump Clean the pump motor cover Replace the pump oil Inspect and clean the inlet filter and clean the inlet filter or replace the gas ballast O ring Clean the pump motor cover the Clean the pump motor cover motor cover Test the motor condition Fit new blades Fit new blades blades Vacuum system Measure down pumping time and final vacuum levels 32 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Maintenance EN Component Maintenance action Vacuum system Measure time to open and close the sample inlet door Clean cover and check operation of fans Air filter Replace filter Ettan MALDI ToF system Perform final test Table 4 3 Preventive maintenance schedule 4 10 Replacing spare parts CAUTION Only use spare parts supplied or specified by Amersham Biosciences In addition to the spare parts for user maintenance the parts listed in Table 4 4 can be replaced by the user Mains
32. oor The slide tray is moved from the target position to the sample entry lock which is then vented to atmosphere This takes about 30 seconds The Vacuum indicator will go off as the vacuum in the sample entry lock is lost In the Instrument Status window Sample is Exchanging will be checked When the door to the sample entry lock has opened remove any sample slide already in the instrument Insert the new sample slide into the sample entry lock Press the slide firmly but gently until it stops Note To minimize moisture in the flight tube do not leave the door open for long periods of time Close the door even if a sample slide is not inserted WARNING Make sure that nobody is working at the sample entry lock when closing the sample inlet door Choose Process Close door to close the sample door The following will happen the closing procedure takes 1 2 minutes e The door closes e The sample entry lock is evacuated by the vacuum system If operational vacuum is not reached within 2 minutes an error message is displayed and the door opens see Chapter 5 for details e The Vacuum indicator is lit e The sample slide is moved into the target position A sound from the sample exchange motor is heard Further operating instructions are provided in the Ettan MALDI ToF User Manual Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 25 ED Basic operation 26 3 3 Closing down the system The instrume
33. ould be performed by the user at regular intervals Interval Instructions reference Daily or when required Clean the instrument Wipe the instrument cover with a soft damp tissue Let the instrument dry completely before use See Section 4 5 1 Check fan operation Check that cooling air flows through the instrument exiting at the rear and top of the instrument See Section 4 6 Monthly Clean the O ring at the sample See Section 4 8 inlet door and check for cracks or other damage Every 6th month Replace the O ring at the See Section 4 8 sample inlet door Table 4 1 User maintenance 28 Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Maintenance EN 4 3 Spare parts CAUTION Only use spare parts supplied or specified by Amersham Biosciences Table 4 2 lists the spare parts that are needed for user maintenance operations Table 4 2 Spare parts required for user maintenance 4 4 Tools and accessories In addition to the spare parts the following tools and accessories are needed for maintenance operations e Gloves powder and lint free e Ethanol 99 5 e Mild detergent e Aluminium foil for covering the sample door opening e A pair of plastic tweezers e int free tissues E Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 29 ED Maintenance 4 5 Cleaning procedures 4 5 1 Cleaning the instrument For proper function the instrument should be kept clean and dry Chemical stains an
34. r is closed 7 The Pirani mbar and Magnetron mbar pressure indications should be decreasing Operational vacuum below 1 x 10 mbar should be reached within 24 hours 8 Before starting an experiment check that the Vacuum indicator lights indicating stable vacuum within specifications 9 During an experiment the Active indicator lights to indicate that data is being transferred between the Ettan MALDI ToF and the controlling computer 3 2 Introducing samples Before introducing samples into the Ettan MALDI ToF instrument they must be purified and incorporated into a suitable matrix that allows the laser beam to desorb and ionize molecules The samples must be dry before introducing into the instrument otherwise the pump down time will be extended General guidelines for sample preparation and examples of specific applications are provided in theEttan MALDI ToF Method Handbook Note Use lint free gloves or a pair of tweezers when handling sample slides The prepared samples should be dispensed into the numbered wells on a sample slide for use with the Ettan MALDI ToF CAUTION Always use sample slides from Amersham Biosciences Avoid depositing materials on the sample slide that may Stick to surfaces of the flight tube e g syrup Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 1 p Basic operation E To introduce samples into the instrument Choose Process Open door to open the sample d
35. re used to enable the rotary pump to evacuate different parts of the system at different stages Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB Reference information qu The turbo pumps comprise the second stage of the vacuum pumping system providing operating vacuum in the flight tube and the reflectron When a sample is to be inserted the inlet chamber is vented to atmospheric pressure When the sample door has closed the inlet valve opens and the backing pump reduces the pressure in the inlet chamber When the inlet chamber pressure has reached about 1 x 107 mbar the sample slide moves into the flight tube and the inlet yalve closes When the pressure in the flight tube has reached 1 4 x 10 mbar the Vacuum indicator lights and an analysis can be started Vacuum chamber Inlet chamber Flight tube Reflectron gauge Turbo pump 2 Turbo pump Vent w Inlet X Backing valve valve 4 valve Turbo pump Turbo pump controller controller S2 S Rotary Pirani pump gauge Vacuum system outlet Fig 6 9 The vacuum system To cover the entire measuring range from atmosphere to operating vacuum two vacuum gauges are used in the vacuum system The Pirani gauge monitors the backing pressure of the rotary pump in the range 1 bar to 10 mbar 0 1 MPa to 0 1 Pa and the magnetron gauge measures the high vacuum range down to 10 mbar 10 Pa For further details on the vacuum components refer to the
36. s Instrument Handbook The protection provided by the instrument may be weakened if other operations are performed WARNING INVISIBLE LASER RADIATION AVOID DIRECT EXPOSURE TO BEAM CLASS 3B LASER PRODUCT Do not open instrument covers Refer servicing to qualified service personnel WARNING HEAVY OBJECT Ihe Ettan MALDI ToF weighs 152 kg 335 Ibs Contact Amersham Biosciences before moving the instrument Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 19 2 Safety instructions 2 4 Built in safety functions Ettan MALDI ToF is certified in accordance with the EN 61 010 1 safety standard To protect the user the instrument also has built in interlocks 2 4 1 Interlocks WARNING Never operate the Ettan MALDI ToF with any interlock disabled 20 High voltage and laser interlocks The high voltage power supplies and the laser are powered by a 24 V DC supply The mains input to this supply is switched on by a relay when a number of conditions are met Table 2 1 lists the interlock conditions that must be fulfilled before the high voltage power supplies and the laser are enabled Interlock condition Type of interlock Magnetron gauge above trip level Hardware magnetron gauge main chamber pressure comparator relay Magnetron gauge fault Hardware magnetron gauge comparator relay Pirani gauge above trip level Hardware pirani gauge backing line pressure comparator relay Reflectron turbo va
37. tes since water molecules from the surrounding air may enter the sample entry lock The desiccant used to dry the vent gas will also be unnecessarily saturated with water Cover the sample inlet door opening with aluminium foil if the door must be open for more than five minutes The sample entry lock 4 8 O ring maintenance CAUTION Damaging the O ring groove may cause vacuum leakage Use plastic tweezers when removing the O ring Take extreme care not to touch the bottom of the groove 1 Place a sheet of aluminium foil on the bench in front of the sample entry door 2 Open the sample inlet door 3 Remove the O ring carefully using a pair of plastic tweezers 4 Check for cracks or other damage to the O ring Replace the O ring if necessary and every 6th month 5 Wipe the O ring with a tissue to remove dust 6 If needed clean the O ring with ethanol 7 Clean the O ring seat and the surrounding area at the sample inlet opening with ethanol 8 Re insert the O ring pressing it into the groove around the sample inlet opening Make sure that it is properly seated in the groove 9 Close the sample inlet door Ettan MALDI ToF Instrument Handbook 18 1144 00 Edition AB 31 ED Maintenance 4 9 Preventive maintenance Table 4 3 lists the preventive maintenance PM operations that should be performed by qualified service personnel during the yearly service visit Ettan MALDI ToF system Perform entry test Connecti
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