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Protein Identification by MALDI Mass Spectrometry Karin Hjernø and Peter Roepstorff Department of Biochemistry and Molecular Biology University of Southern.

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Presentation on theme: "Protein Identification by MALDI Mass Spectrometry Karin Hjernø and Peter Roepstorff Department of Biochemistry and Molecular Biology University of Southern."— Presentation transcript:

1 Protein Identification by MALDI Mass Spectrometry Karin Hjernø and Peter Roepstorff Department of Biochemistry and Molecular Biology University of Southern Denmark Denmark

2 Peptide Mass Fingerprint (PMF) 2) Enzymatic digestion (trypsin) 3) Micropurification ( salt, + concentration) 4) Mass spectrometric analysis, data interpretation. 1) Separation of proteins, here by 2D-gels pI Mw

3 Theoretical Digestion Protein of Interest MALDI-MS...LIHGFYMNKPL......LVCDERTFGHG......HYIGFREWMKL......LIYTSARDEFW.................... Database of known Sequences Experimental Peptide Masses Theoretical Peptide Masses Comparison 679.52 684.34 842.50 856.52 864.48 870.53 1045.57 1126.57 1314.77 1794.87... 1074.50 1087.56 1151.53 1194.60 1200.63 1240.62 1246.57 1275.63 1323.68 1353.73... Protein ID PMF

4 Enzymes for PMF www.matrixscience.com Trypsin High specificity Peptides in a mass range compatible with MALDI Small enzyme

5 MALDI-MS spectrum 1 Da

6 842.5 1602.8 2720.2 Three examples of a normal isotopic distribution at different m/z-value. ? Isotope distribution

7 Removal of contaminants Common contaminants Keratin Tryptic autodigest projects (842.5, 2211.1) Matrix-clusters Overlapping matrix (871.94) and peptide (873.50) peaks.

8 Removal of contaminants Peak Erazor www.welcome.to/ GPMAW

9 Outlier Taking advantage of contaminants! Identification of contaminants Multipoint calibration on contaminants < 20 ppm

10 What!?... No calibrants.. The mass defect: Difference between the monoisotopic and the integer mass value of a given amino acid residue, The mass defect of a peptide around 8-900 Da is.5, around 2000 Da it is.0 (mass dependent) < 50 ppm

11 Filteret and calibrated peak list, what then?!? Make a database dependent search; Open the prefered search program Choose the search database Choose the search parameters Start the search....... Protein candidates

12 Mascot Interface 927.4719 960.5380 999.6130 1026.5727 1055.5372 1066.5641 1072.5767 1088.6055 1099.5488 1129.6679 1199.6724 1211.6559....... 1768.9441 1782.9171 1867.8418 1894.9684 1924.9995 1982.0834 2056.9854 2142.0594 2310.2216 2720.3082

13 Results List 1. gi|6321412 Mass: 76977 Score: 103 gi|6321412 tryptophan synthetase; Trp5p [Saccharomyces cerevisiae] Observed Mr(expt) Mr(calc) Delta Start End Miss Peptide 759.43 758.42 758.41 0.01 695 - 700 0 IGWDLR 830.45 829.44 829.44 -0.00 439 - 445 0 QALNVFR 1 Pyro-glu (N-term Q) 847.47 846.47 846.47 -0.01 439 - 445 0 QALNVFR 907.51 906.50 906.50 0.00 473 - 479 0 FWVTNLK 1003.53 1002.52 1002.57 -0.05 438 - 445 1 RQALNVFR 1031.46 1030.45 1030.48 -0.02 273 - 280 0 DEFFAFQK 1267.72 1266.71 1266.77 -0.06 385 - 396 0 INNALAQVLLAK 1319.64 1318.63 1318.69 -0.05 207 - 219 0 DTPLAVGFGVSTR 1458.70 1457.69 1457.76 -0.07 269 - 280 1 VLSKDEFFAFQK 1484.77 1483.76 1483.79 -0.02 677 - 690 1 GDKDVQSVAEVLPK 1583.75 1582.74 1582.79 -0.05 361 - 373 0 LTEHCQGAQIWLK 1607.80 1606.79 1606.85 -0.05 344 - 357 0 SLYSYIGRPSSLHK 1664.86 1663.85 1663.89 -0.05 620 - 635 0 AQFIAATDAQALLGFK 1702.76 1701.76 1701.79 -0.03 423 - 437 0 FGLTCTVFMGAEDVR ………... Non- significant matches 1 MSEQLRQTFA NAKKENRNAL VTFMTAGYPT VKDTVPILKG FQDGGVDIIE 51 LGMPFSDPIA DGPTIQLSNT VALQNGVTLP QTLEMVSQAR NEGVTVPIIL 101 MGYYNPILNY GEERFIQDAA KAGANGFIIV DLPPEEALKV RNYINDNGLS 151 LIPLVAPSTT DERLELLSHI ADSFVYVVSR MGTTGVQSSV ASDLDELISR 201 VRKYTKDTPL AVGFGVSTRE HFQSVGSVAD GVVIGSKIVT LCGDAPEGKR 251 YDVAKEYVQG ILNGAKHKVL SKDEFFAFQK ESLKSANVKK EILDEFDENH 301 KHPIRFGDFG GQYVPEALHA CLRELEKGFD EAVADPTFWE DFKSLYSYIG 351 RPSSLHKAER LTEHCQGAQI WLKREDLNHT GSHKINNALA QVLLAKRLGK 401 KNVIAETGAG QHGVATATAC AKFGLTCTVF MGAEDVRRQA LNVFRMRILG 451 AKVIAVTNGT KTLRDATSEA FRFWVTNLKT TYYVVGSAIG PHPYPTLVRT 501 FQSVIGKETK EQFAAMNNGK LPDAVVACVG GGSNSTGMFS PFEHDTSVKL 551 LGVEAGGDGV DTKFHSATLT AGRPGVFHGV KTYVLQDSDG QVHDTHSVSA 601 GLDYPGVGPE LAYWKSTGRA QFIAATDAQA LLGFKLLSQL EGIIPALESS 651 HAVYGACELA KTMKPDQHLV INISGRGDKD VQSVAEVLPK LGPKIGWDLR 701 FEEDPSA Sequence Coverage: 51% Significant matches p < 0.05

14 m/z 500 4000 Intensity 18 0 1891.9 2037.0 2191.1 1319.6 1882.0 1475.7 1708.9 2066.0 1307.6 2211.1 1994.0 1393.7 1702.8 1948.0 1435.7 1179.5 2284.2 1607.8 1458.7 1277.7 1638.8 2403.2 2705.2 842.50 1234.6 1073.6 2717.1 2249.1 1045.5 958.46 2754.4 823.56 847.47 759.43 2807.3 578.47 1 MSEQLRQTFA NAKKENRNAL VTFMTAGYPT VKDTVPILKG FQDGGVDIIE 51 LGMPFSDPIA DGPTIQLSNT VALQNGVTLP QTLEMVSQAR NEGVTVPIIL 101 MGYYNPILNY GEERFIQDAA KAGANGFIIV DLPPEEALKV RNYINDNGLS 151 LIPLVAPSTT DERLELLSHI ADSFVYVVSR MGTTGVQSSV ASDLDELISR 201 VRKYTKDTPL AVGFGVSTRE HFQSVGSVAD GVVIGSKIVT LCGDAPEGKR 251 YDVAKEYVQG ILNGAKHKVL SKDEFFAFQK ESLKSANVKK EILDEFDENH 301 KHPIRFGDFG GQYVPEALHA CLRELEKGFD EAVADPTFWE DFKSLYSYIG 351 RPSSLHKAER LTEHCQGAQI WLKREDLNHT GSHKINNALA QVLLAKRLGK 401 KNVIAETGAG QHGVATATAC AKFGLTCTVF MGAEDVRRQA LNVFRMRILG 451 AKVIAVTNGT KTLRDATSEA FRFWVTNLKT TYYVVGSAIG PHPYPTLVRT 501 FQSVIGKETK EQFAAMNNGK LPDAVVACVG GGSNSTGMFS PFEHDTSVKL 551 LGVEAGGDGV DTKFHSATLT AGRPGVFHGV KTYVLQDSDG QVHDTHSVSA 601 GLDYPGVGPE LAYWKSTGRA QFIAATDAQA LLGFKLLSQL EGIIPALESS 651 HAVYGACELA KTMKPDQHLV INISGRGDKD VQSVAEVLPK LGPKIGWDLR 701 FEEDPSA Tryptic auto digest Contaminants

15 1 MSEQLRQTFA NAKKENRNAL VTFMTAGYPT VKDTVPILKG FQDGGVDIIE 51 LGMPFSDPIA DGPTIQLSNT VALQNGVTLP QTLEMVSQAR NEGVTVPIIL 101 MGYYNPILNY GEERFIQDAA KAGANGFIIV DLPPEEALKV RNYINDNGLS 151 LIPLVAPSTT DERLELLSHI ADSFVYVVSR MGTTGVQSSV ASDLDELISR 201 VRKYTKDTPL AVGFGVSTRE HFQSVGSVAD GVVIGSKIVT LCGDAPEGKR 251 YDVAKEYVQG ILNGAKHKVL SKDEFFAFQK ESLKSANVKK EILDEFDENH 301 KHPIRFGDFG GQYVPEALHA CLRELEKGFD EAVADPTFWE DFKSLYSYIG 351 RPSSLHKAER LTEHCQGAQI WLKREDLNHT GSHKINNALA QVLLAKRLGK 401 KNVIAETGAG QHGVATATAC AKFGLTCTVF MGAEDVRRQA LNVFRMRILG 451 AKVIAVTNGT KTLRDATSEA FRFWVTNLKT TYYVVGSAIG PHPYPTLVRT 501 FQSVIGKETK EQFAAMNNGK LPDAVVACVG GGSNSTGMFS PFEHDTSVKL 551 LGVEAGGDGV DTKFHSATLT AGRPGVFHGV KTYVLQDSDG QVHDTHSVSA 601 GLDYPGVGPE LAYWKSTGRA QFIAATDAQA LLGFKLLSQL EGIIPALESS 651 HAVYGACELA KTMKPDQHLV INISGRGDKD VQSVAEVLPK LGPKIGWDLR 701 FEEDPSA ~5500 Da Maldi mass range : from ~ 700 Da to ~ 3500 Suppression effect ( preferential ionization of some components at the expence of others) Post-translational modifications Why not 100% sequence coverage

16 Example – a classical example

17 Manual evaluation – what to look for? Likely/unlikely missed cleavage sites Overlapping peptides Partial modifications Mass accuracy (Intensity of the peaks) ……

18 N C R R RP KR K N C N Missed cleavage sites Digestion using trypsin

19 Missed cleavages Only missed cleavages of one of the following kind are highly likely to be observed (relative to other sites): R/KxxxxxxxR/K xxxxxxxR/KR/K xR/KxxxxxxR/K xxxxE/D R/KxxxxR/K xxxxE/Dx R/KxxxxR/K xxxxR/K E/DxxxxR/K xxxxR/K xE/DxxxxR/K xxxxR/K PxxxxR/K Basic or acidic residue close to the cleavage site in question

20 Digestion using trypsin N C R R RP KR K N C N RxE DR KR

21 Manual evaluation Likely/unlikely missed cleavage sites Overlapping peptides Partial modifications Mass accuracy (Intensity of the peaks) ……

22 Digestion using trypsin N C R R RP KR K N C N DR KR M M ox

23 Oxidation of methionine 16 Da Non-oxidized Oxidized 64 Da -CH 3 SOH Metastable ion SIVPSGASTGVHEALEMRDEDKSK methanesulfenic acid

24 Intensity 12 1811.782 ( 1794.817 ) 1779.793 1783.800 1827.786 m/z 1780 1830 0 0 m/z 11001150 Intensity 700 0 1136.5 1150.61104.6 1119.5 m/z 1250 1300 Intensity 70 0 1282.638 1250.661 1254.644 1266.641 1298.633 +4 +16 +32 +48 Non- modified Partial modification Oxidized tryptophan (W) Oxidized methionine N-terminal pyro- glutamate

25 Manual evaluation – what to look for? Likely/unlikely missed cleavage sites Overlapping peptides Partial modifications Mass accuracy (Intensity of the peaks) ……

26 Example – a classical example

27 Example 2 – a false-positive

28

29

30 Contaminants and calibration

31 PeakErazor

32

33 842.5096 927.4719 960.5380 999.6130 1026.5727 1045.5727 1055.5372 1066.5641 1072.5767 1080.5885 1088.6055 1099.5488 1129.6679 1197.6623 1199.6724 1211.6559....... 1768.9441 1782.9171 1867.8418 1894.9684 1904.0302 1924.9995 1982.0834 2045.0459 2056.9854 2142.0594 2211.0996 2310.2216 2720.3082 2807.2930 Peak List Protein mixtures

34 m/z 500 2500 Intensity 25 0 1361.76 1592.99 1290.69 1446.78 1242.68 1694.88 1450.74 2056.99 1639.95 1567.77 1346.68 1487.74 1467.87 1623.82 1253.65 1768.94 1621.78 1211.66 1088.61 1717.91 1197.66 1782.92 1129.67 1925.00 1687.87 2211.10 1055.54 1099.55 1045.57 2142.061894.97 1072.58 842.510 1080.59 2720.31 2045.05 1904.03 2807.29 960.538 999.613 927.472 2310.22 1026.57 1066.56 1199.67 1439.80 1479.82 1721.91 1738.90 1867.84 1982.08 \\Hermes\prgroup\Karin\Andrea!\Spot # IT 2023 20.9.01 b23753al.massml (11:20 11/02/01) Description: Human Annexin VI Trypsin autodigest Protein mixtures

35

36 m/z 500 2500 Intensity 25 0 1361.76 1592.99 1290.69 1446.78 1242.68 1694.88 1450.74 2056.99 1639.95 1567.77 1346.68 1487.74 1467.87 1623.82 1253.65 1768.94 1621.78 1211.66 1088.61 1717.91 1197.66 1782.92 1129.67 1925.00 1687.87 2211.10 1055.54 1099.55 1045.57 2142.061894.97 1072.58 842.510 1080.59 2720.31 2045.05 1904.03 2807.29 960.538 999.613 927.472 2310.22 1026.57 1066.56 1199.67 1439.80 1479.82 1721.91 1738.90 1867.84 1982.08 \\Hermes\prgroup\Karin\Andrea!\Spot # IT 2023 20.9.01 b23753al.massml (11:20 11/02/01) Description: Human Annexin VI Hypothetical protein XP_038637 (heat shock 70D) Trypsin autodigest Protein mixtures

37 D11 When MS/MS is needed...

38

39

40 Digestion Protein of Interest MALDI-MS (MALDI-) MS/MS R A W G Y V L E Protein ID MS and MS/MS Verification of protein ID, analysis of unassigned peaks

41 Tandem mass spectrometer Mass analyzer Ion SourceDetector Mass analyzer CC Precursor selection Collision Cell, fragmentation of ions Separationof fragments

42 MS of a peptide mixture 899.013

43 MS/MS of a peptide 2+ (collision energy 10 eV) 899.013

44 MS/MS of a Peptide (collision energy 15 eV) 899.013

45 MS/MS of a Peptide (collision energy 18 eV) 899.013

46 Peptide fragmentation Roepstorff and Fohlman, 1984 Biemann, 1988

47 Paizs and Suhai, 2004 Formation of b- and y-ions

48 Identification of the peptide LLQVVEEPQALAAFLR Y1Y1 Y2Y2 Y3Y3 Y4Y4 Y5Y5 Y6Y6 Y7Y7 Y8Y8 Y 10 Y 11 Y 12 Y 13 Y9Y9 899.013 Y 13 EEVV Jens Andersen, Odense, Denmark

49 Manuel Interpretation 115

50 Amino Acid3 LetterCodeSingle Letter CodeResidue Mass Monoisotopic GlycineGlyG57.02147 AlanineAlaA71.03712 SerineSerS87.03203 ProlineProP97.05277 ValineValV99.06842 ThreonineThrT101.04768 CysteineCysC103.00919 IsoleucineIleI113.08407 LeucineLeuL113.08407 AsparagineAsnN114.04293 Aspartic AcidAspD115.02695 GlutamineGlnQ128.05858 LysineLysK128.09497 Glutamic AcidGluE129.04260 MethionineMetM131.04049 HistidineHisH137.05891 PhenylalaninePheF147.06842 ArginineArgR156.10112 TyrosineTyrY163.06333 TryptophanTryW186.07932

51 D Manuel Interpretation

52 D 128 146 Manuel Interpretation

53 Amino Acid3 LetterCodeSingle Letter CodeResidue Mass Monoisotopic GlycineGlyG57.02147 AlanineAlaA71.03712 SerineSerS87.03203 ProlineProP97.05277 ValineValV99.06842 ThreonineThrT101.04768 CysteineCysC103.00919 IsoleucineIleI113.08407 LeucineLeuL113.08407 AsparagineAsnN114.04293 Aspartic AcidAspD115.02695 GlutamineGlnQ128.05858 LysineLysK128.09497 Glutamic AcidGluE129.04260 MethionineMetM131.04049 HistidineHisH137.05891 PhenylalaninePheF147.06842 ArginineArgR156.10112 TyrosineTyrY163.06333 TryptophanTryW186.07932 146??

54 D Q/K Manuel Interpretation

55 D Q/K 172 Manuel Interpretation

56 Amino Acid3 LetterCodeSingle Letter CodeResidue Mass Monoisotopic GlycineGlyG57.02147 AlanineAlaA71.03712 SerineSerS87.03203 ProlineProP97.05277 ValineValV99.06842 ThreonineThrT101.04768 CysteineCysC103.00919 IsoleucineIleI113.08407 LeucineLeuL113.08407 AsparagineAsnN114.04293 Aspartic AcidAspD115.02695 GlutamineGlnQ128.05858 LysineLysK128.09497 Glutamic AcidGluE129.04260 MethionineMetM131.04049 HistidineHisH137.05891 PhenylalaninePheF147.06842 ArginineArgR156.10112 TyrosineTyrY163.06333 TryptophanTryW186.07932 172?!?

57 D Q/K 172 57 Manuel Interpretation

58 Amino Acid3 LetterCodeSingle Letter CodeResidue Mass Monoisotopic GlycineGlyG57.02147 AlanineAlaA71.03712 SerineSerS87.03203 ProlineProP97.05277 ValineValV99.06842 ThreonineThrT101.04768 CysteineCysC103.00919 IsoleucineIleI113.08407 LeucineLeuL113.08407 AsparagineAsnN114.04293 Aspartic AcidAspD115.02695 GlutamineGlnQ128.05858 LysineLysK128.09497 Glutamic AcidGluE129.04260 MethionineMetM131.04049 HistidineHisH137.05891 PhenylalaninePheF147.06842 ArginineArgR156.10112 TyrosineTyrY163.06333 TryptophanTryW186.07932

59 D Q/K G 115 Manuel Interpretation

60 D Q/K G D Manuel Interpretation

61 F D Q/K G D D L Manuel Interpretation

62 F D Q/K G D D L Manuel Interpretation

63 G D G

64 Immonium ions, diagnostic ions

65 K/Q I/L T F K/Q E Immonium ions, diagnostic ions

66 1. Ion series: DGD(Q/K)DFL 2. Ion series: GDG Immonium ions: F,T,(I/L),E,(Q/K)

67 Proline-induced fragmenation (N-terminal to Proline; Xaa|Pro) Most abundant when the Xaa is Val, His, Ile, Leu, Asp Not abundant when Xaa is Pro or Gly Breci, 2003; Kapp, 2003 Fragmentation of VVAASLNPVDFK, singly charged ion

68 Fragmentation of VPTVDVSVVDLTVR, singly charged ion No y13???

69 Peptide with proton predominantly associated with basic residue The proton is mobilized through by collisional activation NH 2 N N N O R1 R2 O R3 O N R4 O B CO 2 H H+H+ NH 2 N N N O R1 R2 O R3 O N R4 O Lys CO 2 H H+H+ Proton mobility If an arginine is present (most basic residue), then the proton is sequestered (non-mobile) and require more energy for mobilization NH 2 N N N O R1 R2 O R3 O N R4 O Arg CO 2 H H+H+ CID or PSD NH 2 N N N O R1 R2 O R3 O N R4 O Arg CO 2 H H+H+ H+H+ If more protons than arginines are present, then a mobile proton will be present MALDIESI

70 Acidic-induced fragmentation Paizs and Suhai Arg Asp Also Glu, but to a lesser degree Charge-remote fragmentation in contrast to Charge-induced fragmentation

71 Fragmentation of VPTVDVSVVDLTVR, singly charged ion y4y9

72 Dominating fragmentation-pathways for singly charged MALDI-peptide ions

73 LSM OX TNDPLEAAR Y6Y6 Y3Y3 Y1Y1 64 Y6Y6 Y3Y3 Y1Y1 Verification of MALDI-TOF/TOF search result

74 Prediction of intense ions

75 The human proteome Isoelectric point (pI) Molecular weight (Mw) Marked spots are differentially expressed between normal red strawberries and a white mutant (half up-regulated and half down-regulated). (fragaria ananassa) Strawberry (not sequenced) Hjernoe et al, 2005, Proteomics, in press

76 Difference in protein expression visual by eye Difference in protein expression detected by the differential analysis software (here DeCyder) Red contra white strawberries Number of spots Proteins found to be down regulated Protein identified based on homology to 1 Flavanone 3- hydroxylaseOnobrychis viciifolia 1 Dihydroflavonol reductaseArabidopsis thaliana 4 O-methyltransferaseFragaria x ananassa 4 Chalcone synthaseFragaria x ananassa.... All four proteins are known to be involved in the flavonoid biosynthesis pathway. One of the functions of flavonoids is to act as a pigment, giving colour to fruits.

77 MS/MS MS Search combining both MS and MS/MS spectra Example of protein identification based on MS/MS spectra from a MALDI-TOF/TOF instrument Search against all green plants

78 Significant hit Details from the search result

79 4-sulfophenyl- isothiocyanate (SPITC) Marakov et al, J. Mass Spectrom. (2003) 38; 373-377 Wang et al., Rapid.Commun. Mass. Spectrom (2004), 18(1); 96-102 SPITC-derivatization, an N-terminal Sulfonation

80 The peptide needs a net- charge of +1 in order to be detected Derivatization of peptides

81 m/z 500 2000 Intensity 10.00 0 + SPITC - SPITC R E AG I T I V Q G D P L E Y N MS/MS Fragmentation of NYELPDGQVITIGAER Found in gi|21538, actin [Solanum tuberosum] 215 Da 1774 1989 Karin Hjernø Fragaria x ananassa MALDI-TOF/TOF spectra obtained at Applied Biosystems 4700 Proteomics Analyzer b2b2 b3b3 y1y1 y1y1 y2y2 y3y3 y4y4 y5y5 y6y6 y7y7 y8y8 y9y9 y 10 y 11 y 12 y 13 y 14 y 15 y 10 y 12 y 13

82 898 920 1051 963 903 905 880 825 834 418 433 455 465 478 487 664 663 708 715 745 780 784 96 118 157 161 212 213 277 281 297 301 324 325 334 349 352 396 397 404 409 416 1112 Chalcone synthase O-methyltransferase Dihydroflavonol reductase Flavanone3-hydroxylase Red 919 1026 899 294 499 509 The Bet v 1-homologous strawberry allergen, Fra a 1 898 920 1051 919 898 920 1051 919 777 Red White Mw pIpI Strawberry allergen-containing spots

83 K* L L T G G H P A S V L SPITC K* L V T G H P A S V L SPITC K* L V T G G H P A S V L SPITC m/z 200 1400 Intensity 0 1392 1435 1449 One spot, three distinct isoforms

84 The End....


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