MS/MS-BASED TECHNIQUES FOR PROTEINS IDENTIFICATIONS Willy - PDF document

MS/MS-BASED TECHNIQUES FOR PROTEINS IDENTIFICATIONS Willy Bienvenut www.unil.ch/paf MS/MS: Introduction and general approach 1

The two main approach for the protein identification including J Proteomic res 2002 , 1 : 239-52 Bottom up approach Top down approach Endoproteinase and cleaving agents Enzyme or Cleaved Cleaving agent Chemical Spécificity residue(s) At the Recurrent C-Term depending Acidic hydrolysis C Low hydrolysis to the conditions W> Y> F>> L Chymotrypsin E Low >M > H C-Term Clostripain E High R >> K C-Term Cyanogen bromide C Very high M C-Term Lysyl endopeptidase E Very high K C-Term N-Halogenosuccinimid C medium W > Y, H C-Term o-Iodosobenzoic acid C High W >> Y C-Term C-Term and N- Pepsin E Very low F, Y, L > E, C Term Prolyl endopeptidase E Low P >> A, S C-Term Prolyl endopeptidase (recombinante) E medium P>>> C-Term Pentafluoropropionic acid C Medium D C-Term Trypsin E High R > K C-Term D and cysteic Peptidyl Asp N E high acid… N-Term S-ethyl trifluorothioacetate C Medium S, T C-Term V8 endopeptidase E High E > D C-Term 2

Ionisation techniques used with MS systems MALDI ESI Matrix-Assisted Laser ElectroSpray Ionisation Desorption/ionisation ESI/MALDI-MS/MS: Qq-TOF 3

Ions separation techniques Resonant ion Quadripolar analyzer MALDI-PSD-MS 4

MALDI- TOF-MS MALDI- TOF/TOF-MS Difference between PSD and CID H,H,a1(+1),a1(+1) 100 1168.4 MALDI-CID-TOF/TOF-MS 90 80 b2(+1),b2(+1) 70 % Intensity 60 50 b4(+1),b4(+1),NTVL(+1) - 18,NTVL(+1) - 18 L,L,I,I b3(+1),b3(+1) 40 b5(+1),b5(+1) b4 - 18(+1),b4 - 18(+1) 30 129.1497 291.2611 b6(+1),b6(+1) b12(+1),b12(+1) b9(+1),b9(+1) 84.1212 a2(+1),a2(+1) 20 366.3346 y7(+1),y7(+1) b5 - 18(+1),b5 - 18(+1) y10(+1),y10(+1) GN(+1),GN(+1) b7(+1),b7(+1) b13(+1),b13(+1) 372.3268 463.3203 77.4003 616.4575 1015.7387 10 174.0723 767.5277 352.2418 493.0316 a9(+1),a9(+1) 81.9112 666.0377 1249.7438 1386.8199 1018.0750 1147.6776 247.1141 757.2271 113.9015 564.2626 873.5460 1261.4395 1371.6366 1155.4902 0 76.0 377.4 678.8 980.2 1281.6 1583.0 Mass (m/z) y15(+1),y15(+1) 100 3322.7 90 MALDI-PSD-MS 80 70 % Intensity 60 50 40 30 20 b9(+1),b9(+1) b12(+1),b12(+1) b14(+1),b14(+1) y7(+1),y7(+1) 10 b10(+1),b10(+1) 77.3781 1386.8404 b6(+1),b6(+1) 772.4402 1137.4435 175.1815 893.2962 1008.8075 1256.7018 93.4102 y2(+1),y2(+1) 492.2276 1452.9201 640.7410 0 76.0 377.4 678.8 980.2 1281.6 1583.0 Mass (m/z) 5

Information from MS data : spot 2 PMF of a Bovine heart ventricle protein, Mr = 43 kDa, pI = 6.6 1675.8021 100 8300.6 90 80 70 % Intensity 60 907.4878 50 40 1157.5495 1231.6365 1643.8131 30 1786.9680 1507.7700 1015.4615 20 1647.8130 1995.9446 950.5359 2152.0556 10 1529.7592 1179.5387 1318.6326 2976.3887 1545.7538 2212.1065 2358.1500 0 882.0 1327.8 1773.6 2219.4 2665.2 3111.0 Mass (m/z) Result : Creatine Kinase Information from MS-MS data MALDI-TOF/TOF MS of the 1507.77 Da peak y 8 b 2 b 4 b 8 100 935.48 313.2 1507.77 LSVEALNSLTGEFK 90 y 12 y 10 y 8 y 6 y 4 80 70 b 4 60 GGDDLDPNYVLSSR % Intensity 50 y 11 y 10 y 8 40 b 2 86.1159 b 4 y 5 30 201.19 b 3 -H 2 O y 7 429.34 b 4 y 8 581.36 y 6 282.30 b 5 20 y 4 345.21 y 10 70.0729 y 11 781.44 y 11 y 9 y 10 500.36 b 8 895.44 694.42 y 12 10 1163.60 60.0540 814.45 1008.49 1079.59 1278.67 1208.62 63.3111 1307.63 133.7069 65.2771 0 49.0 345.2 641.4 937.6 1233.8 1530.0 Mass (m/z) 6

MS-TAG results obtained with the 1507.77 MS/MS spectrum Information from MS data : spot 2 PMF of a Bovine heart ventricle protein, Mr = 43 kDa, pI = 6.6 1675.8021 100 1507.7700 8300.6 100 1825.1 90 90 1508.7741 80 80 70 m/_m = 8000 70 60 % Intensity % Intensity 50 60 907.4878 40 1509.7758 50 30 20 40 1510.7822 1157.5495 1231.6365 10 1511.7819 1643.8131 30 0 1503.0 1505.6 1508.2 1510.8 1513.4 1516.0 1786.9680 Mass (m/z) 1507.7700 1015.4615 20 1647.8130 1995.9446 950.5359 2152.0556 10 1529.7592 1179.5387 1318.6326 2976.3887 1545.7538 2212.1065 2358.1500 0 882.0 1327.8 1773.6 2219.4 2665.2 3111.0 Mass (m/z) Result : Creatine Kinase 7

Information from MS data : spot 2 PMF of a Bovine heart ventricle protein, Mr = 43 kDa, pI = 6.6 1675.8021 100 1231.6365 8300.6 100 4442.7 90 90 m/_m = 14000 80 80 70 70 60 1232.6380 % Intensity % Intensity 50 60 907.4878 40 50 30 1233.6410 20 40 1157.5495 1231.6365 1234.6372 10 1643.8131 30 0 1786.9680 1226.0 1228.6 1231.2 1233.8 1236.4 1239.0 1507.7700 1015.4615 Mass (m/z) 20 1647.8130 1995.9446 950.5359 2152.0556 10 1529.7592 1179.5387 1318.6326 2976.3887 1545.7538 2212.1065 2358.1500 0 882.0 1327.8 1773.6 2219.4 2665.2 3111.0 Mass (m/z) Result : Creatine Kinase ESI-MS/MS : the High-way for complex protein mixture identification 8

Q Trap (Quadrupole – linear trap) The Q-trap MS Protein identification using MS/MS data LC-ESI-MS/MS of a tryptic digest on Q-Trap (PE-Sciex) 9

Peptide fragmentation during MS/MS sequencing From ESI ionisation source 10

Peptide fragmentation during MS/MS sequencing Peptide fragmentation during MALDI-MS/MS sequencing with MALDI ionisation source 100 880.7 HGNTVLTALGGILKK 90 80 b n ions 70 b2(+1),b2(+1) % Intensity 60 50 b4(+1),b4(+1),NTVL(+1) - 18,NTVL(+1) - 18 40 L,L,I,I b5(+1),b5(+1) b3(+1),b3(+1) 30 b4 - 18(+1),b4 - 18(+1) 129.1495 291.2565 b6(+1),b6(+1) b12(+1),b12(+1) b8(+1),b8(+1) 20 a2(+1),a2(+1) 84.1147 437.3590 y10(+1),y10(+1) y7(+1),y7(+1) y2(+1),y2(+1) b13(+1),b13(+1) 10 y6(+1),y6(+1) b8 - 18(+1),b8 - 18(+1) 1015.7315 y13(+1),y13(+1) 0 77.0 378.4 679.8 981.2 1282.6 1584.0 Mass (m/z) 11

Peptide fragmentation during MS/MS sequencing From ESI ionisation source Database interrogation 12

Database interrogation Mascot search-output against SWISS-PROT database N-term side C-term side 13

Monocharged ions 28 Da 18 Da 18 Da 28 Da Peptide fragmentation during MS/MS sequencing 14

MS-Product from Protein prospector: Generate peptides theoretical fragments Peptide fragmentation during MS/MS sequencing From ESI ionisation source C -CAM E E K A C -CAM E I/L T A b 5 -H 2 O Y 10 -H 2 O Y 9 -H 2 O 15

Mascot search-output against SWISS-PROT database Internal fragment Peptide fragmentation during MS/MS sequencing From ESI ionisation source a 2 a 2 -H 2 O C -CAM E E K A C -CAM E I/L T A b 2 -H 2 O x 3 b 2 K Y b 5 -H 2 O Y 10 -H 2 O Y 9 -H 2 O 16

De novo bioinformatic tool approach De novo bioinformatic tool PEAKS: MS/MS de novo Peptide Sequencing (http://www.bioinformaticssolutions.com /software/peaks/index.php) Real sequence : EYEATLEECCAK 17

Peptide fragmentation during MS/MS sequencing From ESI ionisation source De novo bioinformatic tool Real sequence : LVNELTEFAK 18

De novo bioinformatic tool De novo bioinformatic tool vs database search output LKECCDKPLLEK Mascot score: 39 (threshold: 35) 19

De Novo Sequencing approach De Novo Sequencing approach Third computing approach: Research of MS 2 and MS 3 correlation to identified the amino acid sequence 20

Bioinformatic tools for protein identification Database interrogation 21

MS/MS approach • Mascot (public server or on-site server) • MS-FIT (UCSF) • ProFound (PROWL) 22

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