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Performance Characteristics of an AP MALDI Ion Source Applied to Exactive Plus Mass SpectrometerMaciej Bromirski, Olaf Scheibner, Tabiwang N. Arrey, Kerstin StrupatThermo Fisher Scientific GmbH, Bremen, Germany
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IntroductionPowerful Screening and QuantitationTheimportanceofAtmosphericPressureMatrixAssistedLaserDesorption/Ionization(APMALDI)techniqueforanalyzinglargerbiologicalorsyntheticsamplesisinvestigatedonthenovelsinglestageOrbitrapanalyzer,theThermoScientificExactivePlusMS.TheExactivePlusinstrumentallowsaccesstomassrangeuptom/z6000.TheperformanceofthisdeviceispresentedbydetectingMALDI-ionsproducedunderatmosphericpressure.Largerendogenouspeptidesareinvestigatedwithregardtomassresolution,isotopepatternandsignal-to-noiseratio.Intactproteinsarepreparedwith1,5DANmatrixfortheapplicationofIn Source Decay(ISD)fragmentionofproteins;ISDfragmentsofmedium-sizeproteinsareinterpreted.
SamplesarepreparedusingpublishedpreparationprotocolsforMALDIMS.SyntheticpolymerssuchasPEGsareinvestigated,specificallytheperformanceoftheentirepolymerdistributionandtheadductionformationareexplored.
Key WordsExactive Plus, AP MALDI, High Resolution Accurate Mass Perfomance, In Source Decay, Orbitrap Technology
OverviewAmbient Mass Spectrometry: Fast Screening of Large Molecules Novel Aspect: Mass range up to m/z 6000 demonstrated for
AP MALDI MS and Orbitrap™-based technology
Purpose: Compatibility of Thermo Scientific™ Exactive™ Plus MS Instrumentation with focus on singly charged AP MALDI-produced ions at high mass range
Methods: AP MALDI PDF+ ion source attached to Exactive Plus MS
Results: Detection of bovine Insulin at m/z 5734 In Source Decay fragmentation of Cytochrome C Detection of 1 femtomole Angiotensin II with S/N > 50/1 Measurement of synthetic polymer PEG 3350
Methods Exactive Plus MS InstrumentationAllexperimentsareperformedontheExactivePlusmassspectrometer,seeFigure1,usinganAPMALDIPDF+ionsource.TheionsourceiscoupledinfrontoftheheatedcapillarywhichisintegratedintheS-lens.Thepeak-shapedheatedcapillaryisheldatapproximately400-450°Cforallexperimentsshowninthispresentation.Thepeakedfrontendoftheheatedcapillaryfacilitateslaserbeamadjustmentontothesampleplate.TheExactivePlusmassspectrometerisoperatedinFullScanMSandAIFmodesettingsusingresolvingpowerofupto140,000atm/z200(FWHM)andwithmassrangeuptom/z6000.
MassTech’s AP MALDI Source TheAPMALDIPDF+ionsource(MassTech,Inc.,USA),seeFigure2,isusedforthehereinpresentedexperimentsandresults.DistancebetweensampleplateandheatedcapillaryintheMS-interfaceisadaptedwithsuitabledistancepieces(spacers)toadistanceofabout1mm.
ANd:YAGlaserbeamat355nm(pulseduration3-5ns)iscoupledintoa400μmcorediameterfiberformingaspotsizeof500x650μm2.AspiralmotionacrosstheselectedspotontheplatewhilescanningischeckedinMassTech’sTargetSoftware(InstrumentControlSoftwareofAPMALDIsource).Thelaserrepetitionratecanbevariedbetween1and200HzinMassTech’sTargetsoftware.Formostoftheexperimentsrepetitionrateissetto50or200Hz.
Mass Calibration with AP MALDI-Produced Ions in the Exactive Plus InstrumentMasscalibrationoftheOrbitrapdetectorcanbeper-formedinMALDImodeofoperation.UserinterfaceisshowninFigure3(bottom).
Itshowsthenewlyintegratedmasscalibrationeditorforuser-definedmasslists.Specificm/zratioscanbeactivatedordeactivatedfortheindividualmasscalibration.Themasscalibrationisperformedfortheselectedm/zratiosafteranautomatedquickionfluxstabilitycheck.Massstabilityandmassaccuracyarereportedaftermasscalibration.Calibrationprocedureusingtheuser-definedlistlastslessthan1minute.Insetsintosomem/zratiosofMALDICalibrationmixtureMSCAL4aregiven,seeFigure3(top).
Samples and Sample PreparationSigma-Aldrich’sProteoMass™MALDICalibrationKitforLTQXLandLTQHybrids“MSCAL4”(MALDICalibrationmixtures)ispreparedaccordingtotheprotocolprovidedwiththekit.1Alpha-cyano-4-hydroxycinnamicacid(CHCA)matrixisusedtopreparetheMALDIcalibrationsolution(seeFigure3(top)).Ifnototherwisementionedinthefigurecaptions,analytemoleculesaredissolvedinHPLCgradewaterandmixedwithalpha-cyano-4-hydroxycinnamicacid(CHCA)or2,5-dihydroxybenzoicacid(2,5-DHB),respectively.FortheanalysisofIn Source Decayfragmentionsofintact,medium-sizeprotein,1,5diaminonaphthalene(1,5-DAN)matrixisused.2Volumesof0.5-1μLofmatrix-analytemixturesarespottedonastainlesssteelplateandairdried.PolyethyleneGlycol(PEG)ispurchasedfromSigma-Aldrich(P-3640),forsamplepreparationseeresults.
HCD Cell C-Trap Transfer Multipole
S-Lens
AP Maldi
OrbitrapMass Analyzer
Figure 1. Schematic of the Exactive Plus mass spectrometer
Figure 3. User interface (left bottom) with editable mass list and Orbitrap analyzer full scan mass spectrum (top) showing various MSCAL4 peptide signals. Insets show Mellitin pepide at m/z 2845.76145 and ACTH fragment 7-38 at m/z 3657.9289 (exact monoisotopic MH+ values)
FTMS + p MALDI sid=100.00 Full ms [1500.00-4000.00]
2000 2500 3000 3500 4000m/z
1672.9162
1758.9318
1937.9872
2846.7626
3224.85153659.9308
×10
2845.7588R=37026
2846.7626R=38345
2848.7685R=37100
2845 2850 2855m/z
3656 3658 3660 3662 3664m/z
3657.918632708
3659.9308R=32814
3661.9390R=32951
2
Figure 2. MassTech AP MALDI PDF+ source is used for the experiments with Orbitrap-based Thermo Scientific Exactive Plus mass spectrometer
ConclusionFast and broad screening for confident compound ID• Forthefirsttime,intactbovineInsulinatm/z5734is
detectedonanExactiveseriesmassspectrometer.
• In Source DecayfragmentionsofCytochromeCaredetectedwith1,5DANmatrixandN-andC-terminalionsareobserveduptom/z5000.
• S/N>50/1ofa1femtomoleAngiotensindepositedonplateisdetected.
• ForPEG3350,asyntheticpolymer,itspolymerdistributioniseasilydisplayedandadductformationisunambiguouslyelucidatedwith2,5-DHBmatrixandLiTFAaddition.
• AllIonFragmentationallowsdetectionofHCDfragmentionsoverawidemassrange,fromimmonium-relatedionstointactmedium-sizepeptides.
• Itisrecommendedtousehighcapillarytemperatures(about400°C)andselectthespiralmotionforeaseofdataacquisition.
References 1.http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/
Datasheet/2/mscal4dat.Par.0001.File.tmp/mscal4dat.PDForsearchfortheMSCAL4datasheetinhttp://www.sigmaaldrich.com.
2.ThermoScientificApplicationNoteAN30218,MALDIIn Source DecayFragmentIonsofPeptidesandProteinsGeneratedinaCollisionalCoolingInterfaceandDetectedwithanIonTrap–OrbitrapHybridMassAnalyzer
AcknowledgementsWewouldliketothankDr.AndrewHoteling,Bausch&Lomb,USA,forsharingthe2,5-DHB/LiTFAmatrixsamplepreparationprotocolwithus.HelpindatainterpretationofIn Source DecaydataobtainedfromourcolleagueKaiSchefflerisgreatlyappreciated. PresentedattheASMSMeetinginVancouver,2012
5
FIGURE 9. MassTech AP MALDI source attached to the Exactive Plus mass spectrometer
Results Endogenous Peptide and Sequence-Specific Fragments of Medium-Size ProteinBovineInsulinandfragmentionsofintacthorseheartCytochromeCareanalyzedassinglychargedionswiththeExactivePlusMS.
Inthefirstexperiment,theperformancewiththeextendedmassrangeisshownandintactMH+signalofbovineInsulinisdetectedusingCHCAmatrix.Figure4showsOrbitrapanalyzerfullscanspectrumfromm/z1500-6000andinsetsintosingly,doublyandtriplychargedionsofbovineinsulin.Correspondingmassaccuracyandresolvingpoweraregivenintheinsets.Thesameperfor-manceisobtainedusing2,5DHBmatrix,datanotshown.
In Source Decay of Intact Protein Cytochrome CInthesecondexperiment,1,5-diaminonapthalene(1,5-DAN)servesasMALDImatrix,becauseitproducessignificantamountofspecificN-andC-terminalsequenceinformationfromtheIn Source Decayprocess,2Figure5.Seriesofa-,b-,c,y-andz-typeionsareobserved.Here,thez-typeionseriesislabeledinblue.Resolutionandmassaccuracyisgiven.ResultsareinagreementwiththoseobtainedusingotherMALDIinterfaces.2Methodsetupappliesthreemassrangesand75eVInSourceCIDenergy.Aninsetfromm/z1200tom/z5000intoOrbitrapanalyzerfullscaninformation,andaninsetintomassrange(samespectrum)fromm/z2600tom/z3100withsignalassignmentsareshown.
1 femtomol Angiotensin II Amount on the PlateThesignal-to-noiseratio(S/N)ofthemonoisotopicpeakofAngiotensinIIMH+(m/z1046.5418)isevaluated.SamplepreparationwithCHCAmatrixisperformedaccordingtotheprotocolinMSCAL4.TheS/Nofa1femtomoleAngiotensinIIdepositedonthesampleplateisinvestigated.Thelaserrepetitionrateof200Hz,50msinjectiontimeandSourceCIDwereusedfordataacquisition.Atotalof5fullscanswereaveragedperspectrum(Figure6).TheS/Nreflectsthegeneralperfor-manceoflowanalyteloadsonplateinMALDIandAPMALDI.
Figure 4. AP MALDI mass spectrum of bovine insulin at m/z 5734
Figure 5. MALDI In Source Decay of protein Cytochrome C using 1,5 DAN matrix
1500 2000 2500 3000
Minsulin b chain
H+
Minsulin
H+
Minsulin
3H3+
Minsulin
2H2+
3500 4000 4500 5000 5500 6000m/z
100
50
0
Rela
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Abun
danc
e
1911
R=45000
1913
(M+3H)3+, monoisotopic1910.8680, - 3.3 ppm
R=35000
(M+2H)2+, monoisotopic2865.8027, - 1.8 ppm
FTMS + p MALDI sid=100.00 Full ms [1500.00 - 6000.00]
5730 5734 5738
R=20000
(M+H)+, monoisotopic5730.6189, - 1.9 ppm ppm
2866 2868 2870
FTMS + p MALDI sid=75.00 Full ms [600.00-6000.00]
×10
3741.8319
1500 2000 2500 3000 3500 4000 4500m/z
1648.8880R=47376z=1
- 2 ppm
1777.9283R=46712z=1
-2.7 ppm
2008.0764R=44085
3247.5286R=34378
z=1
3133.4846z=1
3375.6248z=1
4024.2632 +2 ppm
4635.2998R=27341z=1
2600 2650 2700 2750 2800 2850 2900 2950 3000 3050
2876.3476R=38349
2675.2284R=40094
2652.4743R=39494 2777.2794 2896.6011
3005.39052834.5817R=38306
3024.6959
all ions are singly charged ionsz=1
2735.51512912.6141
2693.4656
c13
z14
z15
z17
z25
z35
(b- and) c-type ion seriesinclude the covalently bound heme group,this results in a respectively strong mass
defect to (acetylated) N-terminalsequence ions in this protein.
The z-type ion series is labeled in blue.
z23
z25
z26y26
y26
w25w24
c19
c18
c21
c20
x24
m/z
1516.8803R=48964z=1
Rela
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0
10
20
30
40
50
60
70
80
90
100
FTMS + p MALDI sid=25.00 Full ms [800.00-1500.00]
1024.5340R=65103
z=1
1025.5369R=66450
z=1 1044.0998R=71276
z=2
1049.5469R=62153
z=1
1044.0998R=71276
z=2
1048.5453R=64495
z=1
1047.5426R=62663
z=1
1046.5395R=65622
z=1
-2.2 ppm
Rela
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Abun
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5
4
2
3
1
0
1015 1020 1025 1030 1035 1040 1045 1050m/z
1015 1020 1025 1030 1035 1040 1045 1050m/z
Figure 6. Inset into FTMS mass spectrum of a 1 femotomole load of Angiotensin II (m/z 1046.5418, C50 H72 N13 O12) on plate generated by AP MALDI
ResultsAll Ion Fragmentation of Proline14R peptideToshowtheabilityofthe“AllIonFragmentation”(AIF)techniqueintheExactivePlusmassspectrometer,apeptidewiththesequenceProline14R(SigmaAldrich’sProteoMass™P14RMALDI-MSStandard)andMH+ofm/z1533.85765(C76H113N18O16)isinjectedintotheHigherEnergyCollisionalDissociation(HCD)cell.AIFtakesplaceinthecollisioncellattachedtotheC-trap(seeFigure1).FragmentionsaresubsequentlyinjectedintotheOrbitrapdetector;resultsareshowninFigure7.ThespectrumisacquiredusingCHCAmatrix,200Hzlaserrepetitionrate,andappliesSourceCID‘on’.
Synthetic Polymer (PEG) Analyzed with 2,5-DHB Matrix and LiTFAThisexperimentinvolves(2,5-DHBmatrix,theprotocolisgivenasfollows:a)concentrationofpolymersampleis2g/Linmethanol(stocksolution),b)0.25g/LofLiTFA,andc)15g/Lof2,5-DHB,respectively,arepreparedinmethanol.70μLofmatrixsolutionismixedwith10μLofsampleand10μLofLiTFAsolution.1μLofthefinalmixtureisdepositedonthesampleplate.
Additionofe.g.LiTFAinsuchsyntheticpolymersamplescanbebeneficialasitfacilitatesdatainterpretationincomparisontothedefaultoccurrenceofNa+-andK+-ions.TheLi+-adductionformationcancompetesuccessfullyagainstNa+-orK+-adductionformationasshowninthemassspectrumforthePEGsample.Li+-adductionscanbeassignedeasilyanddistinguishedclearlyfromNa+-orK+-adductions.TheexpectedmassincrementofΔm=C2H4O=44.02621ubetweensubsequentpolymerizationgrades(DnandDn+1)isobviousinthemassspectra.
Figure8represents10scansaveragedofthePEGsamplepreparedwith2,5-DHBmatrixandadditionofLiTFA.Orbitrapanalyzerfullscandata(m/z400-m/z6000)(bottom)andaninsetintomassrangearoundm/z2600withpeakinterpretationareshown(top);allmassesarereflectedwithmassaccuracybetterthan3ppm.TheinsetiscomparedtosimulateddataofPEGpolymerizationgradeD60(middle).
Figure 7. FTMS mass spectrum and FTMS/MS spectrum using HCD of a Pro14R peptide
Rela
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Abun
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0102030405060708090
100
FTMS + p MALDI sid=50.00 Full ms2 [113.40-1700.00]
200 400 600 800 1000 1200 1400 1600m/z
6000
20406080
100
Rela
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Abun
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1000 1400 1800m/z
- 1.2 ppm
196.1329R=76382
z=1
227.1750R=73135
z=1
1339.7513R=27953
z=1
1533.8539R=28506
z=1
1549.8472R=28006
z=1
1533.8558R=26757
z=1
1549.8498R=26473
z=1
1145.6460R=31115
z=1
282.2785R=63303
z=1
546.3027R=46093
z=1
740.4079R=39692
z=1
951.5399R=33062
z=1
PPPPPP
PPPP PPPPPP
PPR
4
Figure 8. Polyethylene Glycol (Approx. Molecular Weight 3350, P-3640, Sigma-Aldrich). a) narrow inset of raw data information into mass range m/z 2610 to m/z 2750, peak interpretation and simulation of peak observed around m/z 2667, interpreted as the Li-adduct ion of polymerization state D60 . b) wider inset into full scan information form above with mass range m/z 1000 to m/z 4500
1000 1500 2000 2500 3000 3500
(M(D60(PEG))+Li)+
b)
a)(M(D59(PEG))+Li)+
(M(D59(PEG))+K)+
(M(D59(PEG))+Na)+
(M(D61(PEG))+Li)+(M(D60(PEG))+Li)+
(M(D60(PEG))+K)+
(M(D60(PEG))+Na)+ (M(D61(PEG))+Na)+
(M(D61(PEG))+K)+
4000 4500 4500m/z
m/z
all ions are singly charged ionsz=1
2620 2640 2660 2680 2700 2720 2740
m/z2620 2640 2660 2680 2700 2720 2740
FTMS + p MALDI sid=1.00 Full ms [400.00-6000.00]
2711.63602667.6087
2623.5829
2667.6025
3151.9019R=3666
3372.03472887.7416
3504.11842755.6613
3637.2052R = 33800
2579.5568
1962.1838R=46555
Under the conditions chosen, peaks result in mass resolution
R=39000 at these m/z ratios
Simulation of M(D60
(PEG)+Li)+
C120
H240
O60
Li+H2O
Resolution R=39,000 Charge 1
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www.thermofisher.com/ExactivePlus
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AN30247_E 08/16G
Performance Characteristics ofExactive Plus Instrumentation
Mass range m/z 50-6,000
Resolving Power Up to 140,000 (FWHM) @ m/z 200
Max scan rate Up to 12 Hz at mass resolution setting of 17,500 @ m/z 200
Mass accuracy Internal: < 1 ppm RMS External: < 3 ppm RMS Under defined conditions
Sensitivity In Full MS mode: 500 fg Buspirone on column S/N 100:1 acquired with H-ESI II probe
Dynamic range > 5000:1 in single scan, intra-scan dynamic range
Polarity switching One full cycle in < 1 sec (one full-scan positive ion mode and negative ion mode each at mass resolution setting of 35,000 @ m/z 200)
Analog inputs One analog input (0-1 V) One analog input (0-10 V)
Options• AxialHigherenergyCollision-inducedDissociation
(HCD)cell
• NanoSprayFlexIonsource–singleset-upforallonlinenanoflowapplications
• ESIprobecompatiblewithliquidflowratesof<1μL/minto1mL/minwithoutsplitting
• APCIsourcecompatiblewithliquidflowratesof50μL/minto2mL/minwithoutsplitting
• APCI/APPIsourcecompatiblewithliquidflowratesof50μL/minto2mL/minwithoutsplitting
• Metalneedlekitsforhighandlowflowanalyses
• TheExactivePlusMSisupgradabletoQExactiveMSonsite