MALDI Manual - IMSERC - Northwestern University
Contents
1. Method Information Table Linear Method Constants Approximate Mass IS1 IS2 Lens Delay lt 1600 20 18 55 9 0 Ons 6000 20 18 20 9 0 130ns 9000 20 18 10 9 0 170ns 12K 20 17 90 9 0 190ns 17K 20 17 80 9 0 220ns 25K 20 17 80 9 0 220ns 66K 20 17 50 9 0 240ns Reflector Method Constants Approximate Mass IS1 IS2 Lens Delay lt 1000 19 16 90 9 0 Ons 2000 19 16 85 9 0 80ns 3000 19 16 75 9 0 210ns 6000 19 16 60 9 0 230ns 12K 19 16 35 9 0 360ns 17K 19 16 15 9 0 420ns Initial Starting Method Constants Method File Name IS1 IS2 Lens Refl Refl2 Delay RP_ 0 1kDa 19 16 72 8 3 21 9 7 0 RP_ 0 5k 4kDa 19 16 53 8 49 21 9 7 0 RP 3k 6kDa 19 16 72 8 55 21 9 7 150 LP 0 5k 4kDa 20 18 6 7 0 N A N A 0 LP 2k 20kDa 20 18 5 8 5 N A N A 150 LP 10k 150kDa 20 17 85 9 N A N A 300 LP 30K 300kDa 20 17 86 9 N A N A 500 RAN 0 1kDa 19 16 75 7 5 21 9 7 0 RAN 0 5k 4kDa 19 16 76 8 3 21 9 7 0 RN 2k 10kDa 19 17 1 8 55 21 8 4 250 LN 0 5k 20kDa 20 18 1 8 5 N A N A 150 New Methods vs Old Methods New Methods Old Methods RP_ 0 1kDa par RP 0 5k 4kDa par RP PepMix par RP 0 5k 4kDa AnchorChip par RP PepMix par RP 0 5k 4kDa NALDI par RP NALDI par RP 3k 6kDa par RP ProtMix par RP 3k 6kDa AnchorChip par LP 0 5k 4kDa par LP PepMix par Page 13 of 14 LP 2k 20kDa par LP ProtMix par LP 10k 150kDa par LP 66kDa par LP 30k 300kDa par RAN 0 1kDa par RN 0 5k 4kDa par RN_PepMix par R2. Matrix Suppression lon Source 2 16 72 BA kv 380 IS1 Lens 8 30 gt kv 437 2 2151 Oo eee Deflectiog Reflector 21 00 kv C10 0 Geometry MTP 384 ground steel Reflector 2 9 70 gt kV 2 Rell Carr NO TARGET flexContol RP_PepMix par EE Setect Method Page 9 of 14 Step 8 Select the Processing Tab at the bottom of the screen Ensure that the file name at the bottom is an IMSERC file If it is not then contact the appropriate MS personnel Step 9 Select the Setup Tab at the bottom of the screen Set the Laser Power and the Offset to suitable values for your sample ALWAYS start at a low power and then increase it if needed Step 10 Above where you had previously selected your method select the correct position of your sample Remember the part of the plate that goes into the MALDI first is on the right of the screen Image is rotated clockwise 90 degrees EN flexControl autoflex RP_PepMix par Eile Display View Tools Compass Help Bid GE vee RR Al ut AE N 494 ao r PS EOS PENG Intens 757 33 Sy ye me Vi A ef fab Sum Cursor Peak Characteristics Peak Information 800 m z 757 3265 Peak Width FWHM 0 1193 Resolution 6348 9341 S N Ratio maa Bt S Added 1000 Fteq TCO AA HM kn FARE EG 754 756 758 760 762
3. a Random walk Shots at raster spot 20 x y Speed 10000 10000 um s Relative 5 5 NO_TARGET Absolute 67520 13105 0 Partial sample Complete sample flexControk RP_PepMix par 18 Select Method tof admin Page 10 of 14 Step 11 Above where you had previously selected your sample position select an appropriate number of shots and frequency of shots for your sample Also set the laser offset to approximately 10 15 Note this does NOT turn off the laser PM AU vesels RAN te AJ ati mn PES Intens 757 33 ps arb Sum Cursor 800 2 757 3265 Peak Width FWHM 0 1193 Resolution 6348 9341 S N Ratio C 117 2742 wr AutoXecute Sample Cartier Spectrometer Detection Processing Setup Calibration Status Teaching File Manual Fine Control Advanced gt gt gt Coasire E Auto Teaching el Random walk te vOZZrX T nTMON Shots at raster spot 20 a AE Spot C100 Geomany MTP 384 ground steel v Speed 10000 10000 Relative 5 5 Carie NO_TARGET ad Absolute 57520 13105 State flexControt RAP PepMix par GB Select Method x Help pr Parameter Allowable Values Description Warning Laser Power 0 50 Remember that higher laser power leads to broad and unresolved peaks Shots Any Su
4. 0 set Sum Cursor Peak Characteristics Peak Information m z L 757 3265 Peak Width FWHM 01193 Resolution SAN Ratio X Ceasum GE x Undo e Add EP SaveAs 5 Shots 500 500 Added 1000 Freq 100 0 20 abedo ededolekedelelk veve c Manual Fine Control Advanced gt gt gt Default Teaching Auto Teaching Teach Random walk Shots at raster spot 20 C10 0 Geometry MTP 384 ground steel Speed 10000 Mode Haaie Partial sample NO_TARGET Absolute 4 Complete sample flexControt RP_PepMix par EE Select Method State Step 7 Select the Spectrometer Tab at the bottom of the screen Ensure that Deflection is checked and that the suppress up box is a suitable value for your sample E flexControl autoflex RP_PepMix par MILLE Eie Display View Tools Compass Help Peak Characteristics Peak Information m z Peak Width FWHM Resolution S N Ratio X Clear Sum x Undo de EP SaveAs Freq 10003 AutoXecute Sample Cal etection Processing Setup Calibration Status High Voltage Pulsed lon Extraction Polarity 7 Switched On ar VE m lon Source 1 19 00 ky set relative
5. 62 a EE AutoXecute Sample Carrier Spectrometer Detection Processing Setup Calibration Teaching File Advanced gt gt gt Default Teaching Auto Teaching ex Random walk Teach vOzzrre ronmonwx Shots at raster spot 20 EE EE C100 Geometry MTP 384 ground steel 10000 10200 m s ive 5 5 um NO_TARGET mm solute 67520 13105 um 0 Pattial sample Complete sample flexControt RP_PepMix par JE Select Method Reflector tofadmin x 757 33 y 901 00 DS NN Step 14 You may choose to run additional scans pressing add each time to sum them Otherwise go to File Save As and save your data IMPORTANT DO NOT just press Save This overwrites the prior spectrum even if it is not yours Step 15 Press the Eject Button in the starting screen or the large green button on the side of the MS MALDI Step 16 Remove your sample then insert the plate back into the MS MALDI Make sure for the correct plate loading orientation Step 17 Press the Eject Button in the starting screen or the large green button on the side of the MS MALDI Plate should be inside the MALDI at the end of your session Step 18 Take your sample back to your lab and dispose of it there Page 12 of 14
6. N_ 2k 10kDa par LN_ 0 5k 20kDa par LN_ClinprotMix par LN_PepMix par LN_ProtMix par Tips for MALDI analysis If you observe poor resolution with a linear mode of operation only you must check whether Turbo checkbox is checked or not You can find the option under Detection Tap gt Detector Gain gt Turbo Disable the Turbo checkbox for better resolution This option is only good for MW over 50 kDa enhancing sensitivity of linear detector by compromising resolution We found this option remains enabled regardless of loading a new method if a previous user had enabled This will decrease mass resolution significantly for MW below 10 kDa if enabled For users who analyze proteins above 100 kDa you may have better sensitivity by increasing High Mass Accelerator up to 8 0 kV under Detection Tap gt Detector Gain gt But mass resolution and S N ratio will be significantly decreased as the HMA voltage increases If the baseline increases too high along with increased laser energy change the electronic gain to Regular under Detection gt Electronic Gain gt The default value is Enhanced You may have better signal sensitivity with Highest setting for MW above 10 kDa by sacrificing signal to noise ratio Page 14 of 14 IMSERC MS Personnel Saman Shafaie sepehr northwestern edu Dan Sweeney daniel sweeney northwestern edu Andy Ott a ott northwestern edu
7. Page 1 of 14 MS MALDI User s Manual Update 11 14 2012 Table of Contents EEE RE EE EE EE EE 2 Sample Preparation iese RE es Ed GE Ges ke ee DE es Ge eed N ke ei GE ee Ge Gede Rd EG ee Ge 3 ia Rat CRC dil SA EE RE OE Bruker Autotlex II Operating Procedures nn se ie ds ee EEN ER GN ee ese lai 5 Method Information PM sigs nan audi ne nine 12 IMSERC MS Personnel sesse sees es ee ed ee ee ee ee se ee Ge ee ee ee ee ee AEA ee Ge ee ee ee 14 1 3 Safety General Lab Safety Requirements All users of the IMSERC facility must review the generic IMSERC guidelines before starting training The guideline will be posted by the sign on computer and at http pyrite chem northwestern edu analyticalserviceslab ASL 20Final 20Guideli nes htm Page 2 of 14 Do not run instrument without approval from Saman Jaekuk or lab TA Failure to do so may cause injury damage the instrument produce invalid data and result in additional fees or removal of IMSERC privileges Instrument Specific Safety Hazards Sample to be run Location on equipment PPE required Hazard Mitigation Wear gloves and safety goggles Pinch Hazard Sample Inlet Keep hands clear of sample inlet once you press insert eject button This is especially true if you notice the sample holder is misplaced Page 3 of 14 Sample Preparation 1 Decide if MALDI is the correct technique for your sample 2 Choose the matrix for your s
8. ample Sample Matrix Peptides lt 10kDa a Cyano 4 hydroxycinnanuc acid CHCA Protein gt 10kDa a Sinapinic acid b Super DHB Oligonucleotides a 2 5 Dihydrozybenzoic DHB b 3 Hydroxypicolinic acid c 2 4 6 trihydroxyacetophenone monohydnde THAP polymer a a Cyano 4 hydroxycinnamuc acid CHCA b 2 5 Dihydrozybenzoic DHB c Dithranol Glycosylated protein Super DHB Neutral Carbohydrates 2 5 Dihydrozybenzoic DHB Alpha cyano 50 acetonitrile 0 1 TFA in deionized water Smapimic acid 30 50 acetonitrile 0 1 TFA in deionized water DHB 10 mg ml in water 50 CAN or other appropriate solvent for analyte 3 HPA prepare saturated solution of 3 HPA in Milli Q water 50g L and 50g L solution of diammonium citrate Combine 3 HPA NHacitrate 9 1 Super DHB DHB 10 5 methoxysalicylic acid Preparation Solution A 10 g L DHB in 20 CAN Solution B 10g L 5 methoxysalicylic acid in 50 CAN Combine AB 9 1 3 Obtain an appropriate sample plate Coin Chip to spot your sample on Coin Chips are available from IMSERC staff for 35 Plates with pre applied matrix with calibrant spots AnchorChip and matrix free plates NALDI are available cost is 100 plate or 1 site 4 Make a solution of approximately 10 sample to matrix by mass disregard solvent for this calculation 5 Apply solution to plates 1ul per spot 6 Dry the sample before coming to IMSERC solvent must be evaporated 1 Page 4
9. m shots to build signal with lower power multiple spectra can be added Frequency 0 200 Use low frequencies to move around and see changes Sample Shots spot If sample is being depleted use Carrier Mode on partial random walk to automatically move Random off to a new spot walk Spectrometer Do not change Settings are optimized for selected mass ranges Detection Use default Window Zoom in later Before any parameter is set outside the limits in this table approval must be obtained from IMSERC staff members Failure to do so may cause damage to the instrument produce invalid data and result in additional fees or removal of IMSERC privileges Page 11 of 14 Step 12 Press start Move the crosshairs around the sample left click with mouse to find a good spot You may also need to change the aforementioned settings Note if you are getting a hump near the left of the spectrum you are likely using too much laser power Step 13 After obtaining a satisfactory spectrum press add EN flexControl autoflex RP_PepMix par Bile Display View Tools Compass Help Bid AE Oo ex BAAR AR AA dt d HOE OG Da ST Re re AIS SN 100 Intens arb Sum Cursor Peak Characteristics Peak Information m z 757 3265 Peak Width FWHM 0 1193 Resolution 6348 9341 S N Ratio 117 2742 AAR A ter a d RE SD ME OE IR PG F 18 21 2 M 754 756 758 760 7
10. of 14 Pre Run Checklist Step Instruction Comments Verify sample is completely dry on the sample plate Poor vacuum poor signal and resolution as well as long transfer times will result from wet samples 2 Log on to instrument in Login system 3 If needed log into Login Name tof user computer Login Password youshouldknow 3 Start Flex Control Log in as tof user you don t need to do anything No password is required 4 Check status lights on Mains Green instrument panel System Ready or Warm up Target Access Page 5 of 14 Bruker Autoflex III Operating Procedures Northwestern University Analytical Services Laboratory Last Updated 8 8 2008 by AWO Starting Screen El fhexControt autoflex RP PepMix par Ble rele View Tacks Compass Help PE GW 0 eogi p Multi Pane View Peak labeling options BOSE 1094 oo TM AA RAR AA TG ET Samle Came Spechometer Detection Pracesang Setup Caibrabon Statue Teacheg Fie MTP 384 ground steel Manual Fee Coral Raster Scan clickon advanced to see Desa Teaching mo ANN num ter sak danset 3 a LA 5 i i 200 Method Editor Screen Step 1 Press the Eject Button in the starting screen or the large green button on the side of the MS MALDI The Tray will open slowly Make sure the tray path is not obstructed Step 2 Load the Sample Step Instruction Commen
11. ts 1 Remove Always store sample carrier in instrument to ensure that the sample plate next person can find it Page 6 of 14 Place plate Sample coin chip is keyed to only fit one way Ensure plate on carrier sits flush with the top of the carrier and sample plate is flat Wrong Sample may scratch sealing surface and cause a leak Load Plate Right Carrier flush against back of transfer system into Loading Dock Page 7 of 14 Wrong Carrier two far forward Sealing surface can be scratched Wrong Carrier Backward Error occurs eraso Step 3 Press the Eject Button in the starting screen or the large green button on the side of the MS MALDI to insert your sample Step 4 While sample is loading click Select Method button from the starting screen Select the method appropriate for your sample The naming convention is for basic operation RP 0 5k 4kDa par First character TOF mode L linear R Reflectron Second Character polarity P positive N Negative Third phrase optimized and calibrated molecular weight range Step 5 Press Open to choose your method Page 8 of 14 Step 6 Select the Sample Carrier Tab at the bottom of the screen Ensure that Off is checked for Mode E flexControl autoflex RP PepMix par file Display View Tools Compass Help Bid AU osse amp RAS Al ud MOEDER pr Jn 2 757 33 5 10
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