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

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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

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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

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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

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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

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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

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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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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