布鲁克MALDI-TOF MS 入门小册子

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InstructionsforUse

StarterKitforMALDI-TOFMS

Forcalibrationandtestingofmatrix-assistedlaserdesorptionandionizationtime-of-flightmassspectrometers(MALDI-TOFMS)

Productdescription

ThekitprovidesarangeofstandardpeptidesandproteinsforthepurposeofcalibrationandtestingofMALDI-TOFmassspectrometers.Thiskitisdesignedtoprovidestandardsformostproteinandpeptideapplications.Recrystallizedmatrices(tube1-3)withhighpurityaresupplied.Acombinationofpeptides(tube4)providesgoodcalibrationacrossatypicalmolecularmassrangebetween~1000and3500Da.Twomixturesofproteins(tube5+6)allowinstrumenttestingandcalibrationsforthemolecularmassrangebetween~3000and70000Da.

Aspecialmixtureofdifferentpolypeptidechainfragments(tube7)wasproducedbythetrypticdigestofbovineserumalbumin.Threesinglepeptides(tube8-10)areprovidedforadjustmentofthemassspectrometerandcompletethekit.

AllofthestandardsandmatricesinthekitmayalsobepurchasedasindividualcomponentsusingthepartnumberlistedintheOrderingInformationpartattheendofthisdocument.Thecompo-nentswereinvestigatedandtestedonaBrukerDaltonicsMALDI-TOFmassspectrometer.

Kitcomponents

Tube

Substance

Amount

Matrices

1a-Cyano-4-hydroxycinnamicacid(HCCA)200mg

2Sinapinicacid(SA)200mg32,5-Dihydroxybenzoicacid(2,5-DHB)200mg

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Tube

Substance

Amount

Peptide/proteinstandards

4Peptidecalibrationstandard

5ProteincalibrationstandardI

6ProteinstandardII

7Trypticdigestofbovineserumalbumin

Peptides

8AngiotensinIl

9ACTH18-39

10Somatostatin28

for250calibration

for250calibrationfor250spotsfor250spots

500pmo

500pmol

500pmol

spots

spots

Storageandstability

Thekitisshippedatambienttemperatures.Werecommendstoringallcomponentsonarrivalatlessthan0℃.Samplesolutionsshouldbealiquotedandfrozen.Wedonotrecommendrefreezingsamplesafterthawing.

Riskandsafetyinformation

ThekithastobelabeledaccordingtoRegulation(EC)No1272/2008.Itcontainscomponentsthatareclassifiedashazardouschemical(Signalwords:DANGERandWARNING).PleasereadtheMaterialSafetyDataSheetwhichisavailablefordownloadat/msdsintheproductdescriptionarea.

Besidethekitcomponents,werecommendfurtherchemicalswithintheseInstructionsforUse.PleasereadandobservetherespectiveMaterialSafetyDataSheettobeprovidedbyyoursupplier.Observethegeneralsafetyregulationswhenhandlingchemicals.

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Characterizationandmeasurementresultsofpeptidecalibrationstandard

Thenexttablecontainsmolecularweightsofallcomponentsinthepeptidemixtures(peptidecalibrationstandard).Fig.1showsresultsofmeasurement.

Peptide[M+H]+Monoisotopic[M+H]Average

AngiotensinIl

AngiotensinI

SubstanceP

Bombesin

ACTHclip1-17

ACTHclip18-39

Somatostatin28

1046.5418

1296.6848

1347.7354

1619.8223

2093.0862

2465.1983

3147.4710

1047.19

1297.49

1348.64

1620.86

2094.43

2466.68

3149.57

Fig.1:MALDImassspectrumofpeptidecalibrationstandard.TargetpreparationwithHCCAmatrix.

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Characterizationandmeasurementresultsofproteincalibrationstandards

Thekitcontainstwodifferentproteinmixtures(IandIl)astestandcalibrationstandardswithfollowingcompositions.

ProteinstandardIAveragem/zProteinstandardIlAveragem/z

Insulin[M+H]

5734.51

Trypsinogen[M+H]*

23982

Ubiquitin[M+H]+

8565.76

ProteinA[M+H]+

44613

CytochromeC[M+H]+

12360.97

Albumin-Bovine[M+H]

66431*

Myoglobin[M+H]+

16952.30

ProteinA[M+2H]²

22306

CytochromeC[M+2H]²·Myoglobin[M+2H]²+

6180.99

8476.65

Albumin-Bovine[M+2H]²+

33216*

Bovineserumalbuminisnotrecommendedforcalibrationpurposesduetoamassshifttohigherm/zvaluesbyunspecificadductformation(s.Fig.4).Ifaninstrumentcalibrationinahigherm/zrangeisrequired,weadvisean

extrapolationofalinearcalibrationbasedonthesignalsatm/z22306,23982and44613.

Thenexttwofigures2and3showdiferentspectraofproteincalibrationstandardIduetodifferentpreparationsofMALDI-TOFtargets.

Fig.4showsthespectrumofproteincalibrationstandardII.ThetargetwaspreloadedwithSA.

17000m/z

Fig.2:MALDImassspectrumofproteincalibrationstandardI.Targetpreparation

withHCCAmatrix.

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Fig.3:MALDImassspectrumofproteincalibrationstandardI.Targetpreparation

withSAmatrix.

Fig.4:MALDImassspectrumofproteincalibrationstandardII.Targetpreparation

withSAmatrix,linearcalibrationwithproteinA(1-and2-foldcharged)and

trypsinogen(1-foldcharged).

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Characterizationandmeasurementresultsoftrypticdigestofbovineserumalbumin

Differentproteolyticpolypeptidescharacterizethetrypticdigestofbovineserumalbumin(BSA).Inthenexttablethedifferentpeptidesandmolecularmassesarelisted.Figure5depictsatypicalMALDI-TOFspectrumoftheBSAdigest.

Fragments

Intactprotein

[161-167]

66-75]

[361-371]

402-412]

[569-580]

360-371]

[421-433]

347-359]

[437-451]

[347-360

508-523]

529-544]

168-183]

[M+H]+Monoisotopic

927.493

1163.631

1283.711

1305.716

1399.693

1439.812

1479.795

1567.743

1639.938

1723.844

1880.921

1907.921

2045.028

[M+H]+Average

66431.00

928.06

1164.33

1284.49

1306.49

1400.62

1440.67

1480.69

1568.71

1640.90

1724.89

1882.13

1909.15

2046.31

Fig.5:MALDImassspectrumoftrypticdigestofbovineserumalbumin.Anchor-Chip"MtargetpreparationwithHCCAmatrix.

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Samplepreparationprocedure(recommendation)

Pre-Remarks

Poorsamplepreparationwilldegradesensitivity,yieldlowresolutionandpoorreproducibility.ThegenerationofionsthroughMALDIdependsontheproductionofasuitablecompositematerial,consistingofthematrixsubstanceandtheanalyte.Forbestresultsuseonlychemicalsofhighestavailablepurity.

Chemicalsandmaterialsrequired

Acetonitrile(ACN)

0.1%Trifluoroaceticacid(0.1%TFA)inwater

:Acetonitrileisclassifiedashazardouschemical(DANGER:H:225,319,302,312,332;P:210,280,305+351+338).

Necessaryequipmentandtools:centrifuge,shaker,ultrasonicdevice,pipettes,pipettetips

1.Preparationofsamplesolution

Dissolvesamples(wholecontentsoftubes4-10)ineachcasein125μL0.1%TFAsolventandshakeforseveralseconds.

2.Preparationofmatrixsolution

ProduceamixofACNand0.1%TFAinavolumeratio1:2.SolveHCCA,SAor2,5-DHBinthis

solventuptosaturationatroomtemperature.Assistthesolutionprocessbyhelpofultrasonic

device.Spindownexcessmatrixinacentrifuge(5minat14000rpm)anduseonlythe

homogeneoustransparentphase.Usematrixsolutionsaccordingourrecommendationsinthe

followingtable.

Tube

Sample

Matrix

PeptidecalibrationstandardHCCA,2,5-DHB

5ProteincalibrationstandardIHCCA,SA,2,5-DHB

6ProteincalibrationstandardIlSA,2,5-DHB

7TrypticdigestofbovineserumalbuminHCCA,2,5-DHB

8AngiotensinIlHCCA,2,5-DHB

9ACTH,clip18-39HCCA,2,5-DHB

10Somatostatin28HCCA,2,5-DHB

3.PreparationontoMALDI-TOF-Target

Accordingtothedrieddropletmethod,mixequalvolumesofsampleandmatrixsolution(HCCA,SAor2,5-DHB).Apply1μLontoastandardsteeltargetanddryatroomtemperature.

ForAnchorChip'Mtargetpreparation,refertotheAnchorChipInstructionsforUse(#8215344).

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OrderingInformation