lab 1 - klinisk kemisk diagnoskbme.lth.se/.../lab1_presentation_mr_vs2.pdf · 2017. 12. 11. ·...

MassSpectrometry

Quan%fica%on&Proteiniden%fica%on

bypep%demassfingerprint

[email protected]

Lab1-KliniskKemiskDiagnos9k

•  MALDImassspectrometry•  Quan%fica%onbyMALDIMS•  Proteiniden%fica%onbypep%demassfingerprint(PMF)

Lab1-KliniskKemiskDiagnos9k

Mee%ngroom

MSlab

Sampleprepara%onSAFETY:Labcoatandsafetyglasses

•  Massspectrometryisananaly%caltechniqueusedtomeasurethemass-to-chargera-oofions.

•  Thisisachievedbyionizingthesampleandsepara-ngionsofdifferingmassesandrecordingtheirrela%veabundancebymeasuringintensi%esofionflux.Atypicalmassspectrometercomprisesthreeparts,anionsource,amassanalyzer,andadetectorsystem:

MassSpectrometry

Karas&HillenkampAnal.Chem.198860,2299-2301.Fennetal.Science1989,246,64-71

• Sample in liquid

• Purification is needed

• Sample in solid state • Purification optional

MALDI-Matrixassistedlaserdesorp%on/ioniza%on

ESI-Electrosprayioniza%on

Ioniza9onofbiomolecules

MALDI-LTQOrbitrapXL

zmk/

=ω

w =oscilla%onfrequencyk =instrumentalconst.m/z=….whatwewant!

OrbitrapFTmassanalyzer

Quan9fica9onStandardcurve

Concentration!pmol/µl!

Mean! (Height)!

Stdev!(Height)!

RSD! (%)!

2.5" 2267" 728" 32.1"

2.0" 2059" 732" 35.5"

1.5" 1067" 312" 29.3"

1.0" 354" 81" 22.7"

0.5" 122" 54" 44.5"

RSD<45%

StandardcurvewithoutIS

Quan9fica9onbyusingstableisotopelabeledstandards

PAR=Light(Analyte)PeakAreaHeavy(SIS)PeakArea

HLMS

Analyte SIS

SIS:StableIsotopeStandardPAR:PeakAreaRa%o

•  pep%deswith13Cand/or15Nmodifiedaminoacids(arginineorlysine)

•  chemicallyiden%caltothetargetpep%debutwithmassdifference(similarbehaviortothetargetpep%dewithregardstochromatographicsepara%on,ioniza%on,andfragmenta%on)

Analyteconcentra%on=PAR*SISpep%deconcentra%on

Concentra9onpmol/µl!

Mean(HA/HIS)!

Stdev(HA/HIS)!

RSD(%)!

2.5" 0.549" 0.017" 3.1"

2.0" 0.453" 0.027" 6.1"

1.5" 0.338" 0.027" 7.9"

1.0" 0.231" 0.038" 16.6"

0.5" 0.142" 0.0005" 0.3"

RSD<17%

StandardcurvewithIS

Task1–Genera%onofastandardcurve

10uL10uL

10uL

10uL10uL

10uL10uL

10uL10uL

10uL10uL

lightstock 2x 4x

8x16x

32x

1.  Preparea2folddilu%onseriesofsample1(lightpep%de)in5steps2.  Preparematrixsolu%onwithSIS(heavypep%de).3.  Add1uLsample+1uLmatrix(withSIS)solu%ononaMALDItarget

plate(MTP).4.  CollectMSdataandtransferpeaklisttoUSBs%ck.5.  CreatestandardcurveinExcelwithnormaliza%ontoSISpep%de

2uLSISstock

38uLmatrixsolu%on

+/-1Da

+/-2Da

MW

+/-0.02Da

1kDa 20kDa 60kDa

Moresampleisneeded

accuracy

MSisasophis%catedbalanceformanyanalytesincludingproteins

Belersensi%vityandaccuracy Moresampleisneeded

Proteiniden9fica9onMoredifficulttomeasurelargethings

Na%veprotein

8MUREA

Proteindiges%onwithspecificaenzyme

reduc%on/alkyla%on(op%onal)

enzyme(trypsin)

pep%des

1 66

173

25

184

198 195192

171170

31

3446

48 50

219

69 71

151

78

138 126

110108

207 209202

234246255 252251

173

25

184

198 195

171

31 6648 50

219

151 138

207 209

234

Noreduc%onresultsinlongbranchedpep%des!4164Da7434Da

2230Da

Importanceofreduc%on/alkyla%on–Disulfidebridges

•  Non-reduced

[email protected]:2574.0Laser: 5.0

28-Apr-2008PSA digest Sigma G2

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900m/z0

100

%

AV_20080428_01 4 (0.130) Cm (1:5) TOF LD+ 1.15e41450.19;11495

695.691946

696.71926 782.20

747

1451.2210246

2292.217038

2291.185616

1452.254936

1766.173911

1453.291586

1454.29819

1926.203843

1768.242252

1769.261056 1909.97

876

1928.242354

1929.281213

1968.22694 2132.29

565

2293.245389

2294.273214

2295.301655

2296.30943

1407.1

1872.1

2229.2

[email protected]:2574.0Laser: 5.0

30-Apr-2008PSA Sigma digest H2

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900m/z0

100

%

AV_20080430_02 1 (0.030) Cm (1:5) TOF LD+ 9.12e31407.81;9123

854.342950

655.911663

757.361363

673.221052

674.24570

758.36588

1077.551717

855.341421

1006.43774

856.34665

1078.541034

1079.56559 1272.72

548

1408.817293

1439.805479

1440.794375

1872.013171

1715.892733

1441.812058

1442.80796

1716.892392

1717.921487

1856.15791

1718.91748

1874.031796

2229.2312581875.01

1004

1888.00636

2377.301058

2261.231057

2344.32669

2589.44952

2379.32593

2591.44563

1272.7

1077.5

854.3

757.4

674.2

1872.1

2229.2 2344.3 2589.4

•  Reduced

MALDI-MS

Na%veprotein

8MUREA

reduc%on/alkyla%on(op%onal)

enzyme(trypsin)

pep%des

MS

MSanalysisdeterminestheweightofindividualpep%des

MSanalysisoftheproteoly%cpep%des

Na%veprotein

8MUREA

enzyme(trypsin)

pep%des

Proteiniden%fica%onusingPMF

MS

MS

Databasesearch

ExperimentaldeterminedTheore%calDetermined

Pep%demassfingerprin%ng=PMF

•  Simpleandpowerfullanalysismethod.•  Proteiniden%tycanbeobtainedfrom”clean”samples.•  PaXernisrecognizednotthesinglepep9des!

PMFfrom3differentproteins

Pep%demassfingerprint

1000 1200 1400 1600 1800 2000Mass(m/z)

6.0E+4

0102030405060708090

100%In

tensity

4700ReflectorSpec#1[BP=1267.6,60397]

1267

.599

0

1759

.814

5

1384

.603

513

37.568

412

51.596

8

1641

.731

2

1195

.570

3

1951

.803

8

1098

.497

3

1871

.849

918

57.608

5

1736

.803

8

1367

.564

9

1633

.764

0

1137

.454

8

1013

.419

9

1979

.943

0

1246

.522

1

1660

.654

1

1159

.541

011

18.505

711

93.433

212

72.611

8

1608

.720

315

80.694

5

1032

.484

9

1506

.661

515

66.380

5

1958

.852

8

1753

.789

8

1283

.608

2

1832

.703

9

1439

.605

2

1897

.701

2

1494

.711

5

1320

.549

213

03.579

1

1090

.486

6

1927

.553

7

1221

.577

1

1814

.713

9

1351

.653

1

1775

.781

9

1456

.615

415

23.659

114

79.697

6

1594

.579

3

1412

.618

2

1045

.479

0

1708

.759

6

1000 1200 1400 1600 1800 2000Mass(m/z)

9.2E+3

0102030405060708090

100

%In

tensity


1479

.606

014

39.625

5

1567

.542

816

39.731

8

1283

.540

0

1823

.667

0

1305

.537

5

1022

.276

9

1537

.606

4

1423

.311

0

1888

.730

2

1163

.468

5

1050

.259

4

1347

.328

4

1667

.601

7

1511

.655

8

1723

.606

9

1249

.439

112

88.172

2

1443

.226

713

67.413

1

1569

.152

7

1461

.601

313

86.412

713

99.327

1

1851

.662

717

89.667

6

1597

.559

9

1873

.477

519

40.556

3

1327

.453

1

1964

.712

2

1116

.380

1

1493

.568

2

1011

.265

0

1646

.231

215

80.580

1

1141

.279

411

85.418

9

1703

.618

3

1549

.540

4

1000 1200 1400 1600 1800 2000Mass(m/z)

7.6E+4

0102030405060708090

100

%In

tensity


1168

.479

0

1296

.544

1

1633

.409

916

06.712

5

1478

.627

314

70.477

1

1598

.562

9

1734

.801

1

1350

.540

5

1125

.317

0

1433

.592

3

1631

.406

9

1152

.135

1

1712

.601

6

1172

.720

8

1456

.469

8

1105

.375

2

1840

.679

4

1495

.489

115

61.681

615

84.553

3

1280

.228

5

1616

.159

716

49.401

6

1301

.545

2

1092

.455

211

36.427

611

89.388

812

30.387

113

13.477

2

1040

.827

0

1339

.472

7

1698

.577

5

1067

.413

7

1826

.667

7

Start-End Observed Mr(expt) Mr(calc) ppm Miss Sequence! 1 - 9 1077.5035 1076.4962 1076.4961 0 0 -.IVGGWECEK.H! 10 - 21 1407.7516 1406.7443 1406.7419 2 0 K.HSQPWQVLVASR.G! 24 – 44 2344.2219 2343.2147 2343.2093 2 0 R.AVCGGVLVHPQWVLTAAHCIR.N! 47 - 53 757.4924 756.4851 756.4858 -1 0 K.SVILLGR.H! 54 – 83 3493.6915 3492.6842 3492.6925 -2 0 R.HSLFHPEDTGQVFQVSHSFPHPLYDMSLLK.N! 54 - 83 3509.6924 3508.6852 3508.6874 -1 0 R.HSLFHPEDTGQVFQVSHSFPHPLYDMSLLK.N Oxidation (M)! 86 - 101 1871.9475 1870.9403 1870.9360 2 0 R.FLRPGDDSSHDLMLLR.L! 86 - 101 1887.9422 1886.9349 1886.9309 2 0 R.FLRPGDDSSHDLMLLR.L Oxidation (M)! 102 - 113 1272.6695 1271.6623 1271.6609 1 0 R.LSEPAELTDAVK.V! 114 - 145 3540.6517 3539.6444 3539.6476 -1 0 K.VMDLPTQEPALGTTCYASGWGSIEPEEFLTPK.K Oxidation (M)! 146 - 167 2588.3109 2587.3036 2587.2999 1 1 K.KLQCVDLHVISNDVCAQVHPQK.V! 171 - 177 854.4007 853.3934 853.3939 -1 0 K.FMLCAGR.W! 171 – 177 870.3958 869.3886 869.3888 -0 0 K.FMLCAGR.W Oxidation (M)! 222 - 226 673.3768 672.3695 672.3707 -2 0 K.VVHYR.K

72%sequencecoverage! 1 IVGGWECEKH SQPWQVLVAS RGRAVCGGVL VHPQWVLTAA HCIRNKSVIL! 51 LGRHSLFHPE DTGQVFQVSH SFPHPLYDMS LLKNRFLRPG DDSSHDLMLL!101 RLSEPAELTD AVKVMDLPTQ EPALGTTCYA SGWGSIEPEE FLTPKKLQCV!151 DLHVISNDVC AQVHPQKVTK FMLCAGRWTG GKSTCSGDSG GPLVCNGVLQ!201 GITSWGSEPC ALPERPSLYT KVVHYRKWIK DTIVANP

Databasesearchresult(MASCOT)

Score:94

Task2–Iden%fica%onofunknownproteinsbyPep%deMassFingerprint

(PMF)

1.  Select3differentunknownproteinsamples(readydigests).2.  Add1uLsample+1uLmatrixsolu%ononaMALDItarget

plate(MTP).3.  CollectMSdataandtransferpeaklisttoUSBs%ck.4.  PerformdatabasesearchusingmascotPMF

hlp://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF

1.  Matrixmixedwiththeheavypep%destandard

2.  Samplewithunknown

concentra%on(1sample)

1.  Dilu%onseries(5dil.steps)2.  Samplewithunknown

concentra%on(1sample)

Task1(Pep%dequan%fica%on)

10uL10uL

10uL

10uL10uL

10uL10uL

10uL10uL

10uL10uL

lightstock 2x 4x

8x16x

32x

2uLSISstock

38uLmatrixsolu:on

10uL10uL

10uL

10uL10uL

10uL10uL

10uL10uL

10uL10uL

lightstock 2x 4x

8x16x

32x

2uLSISstock

38uLmatrixsolu:on

Task2(Pep%deMassFingerprint)

1.  Unknownproteindigests(3samples)

1.  Unknownproteindigests(3samples)

1a.Calibra%oncurvegenera%on1b.Calcula%onoftheunknownconcentra%onof

thesamples(1sample/group)

2.  Databasesearch,iden%fica%onoftheunknownproteins(3samples/group)

lab 1 - klinisk kemisk diagnoskbme.lth.se/.../lab1_presentation_mr_vs2.pdf · 2017. 12. 11. ·...

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