MS/MS-BASED TECHNIQUES FOR PROTEINS IDENTIFICATIONS
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1 MS/MS-BASED TECHNIQUES FOR PROTEINS IDENTIFICATIONS Willy Bienvenut MS/MS: Introduction and general approach 1
2 The two main approach for the protein identification including J Proteomic res 2002, 1: Bottom up approach Top down approach Endoproteinase and cleaving agents Clostripain Pepsin Trypsin Cleaving agent Acidic hydrolysis Chymotrypsin Cyanogen bromide Lysyl endopeptidase N-Halogenosuccinimid o-iodosobenzoic acid Prolyl endopeptidase Prolyl endopeptidase (recombinante) Pentafluoropropionic acid S-ethyl Peptidyl Asp N trifluorothioacetate V8 endopeptidase Enzyme or Chemical C E E C E C C E E E C E E C E Spécificity Low Low High Very high Very high medium High Very low Low medium Medium High high Medium High Cleaved residue(s) Recurrent hydrolysis W> Y> F>> L >M > H R >> K M K W > Y, H W >> Y F, Y, L > E, C P >> A, S P>>> D R > K D and cysteic acid S, T E > D At the C-Term depending to the conditions C-Term C-Term C-Term C-Term C-Term C-Term C-Term and N- Term C-Term C-Term C-Term C-Term N-Term C-Term C-Term 2
3 Ionisation techniques used with MS systems MALDI Matrix-Assisted Laser Desorption/ionisation ESI ElectroSpray Ionisation ESI/MALDI-MS/MS: Qq-TOF 3
4 Ions separation techniques Resonant ion Quadripolar analyzer MALDI-PSD-MS 4
5 MALDI- TOF-MS MALDI- TOF/TOF-MS Difference between PSD and CID TOF TOF MS/MS Precursor Spec #1[BP = 110.1, 1168] H,H,a1(+1),a1(+1) b2(+1),b2(+1) MALDI-CID-TOF/TOF-MS % Intensity 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) b6(+1),b6(+1) b12(+1),b12(+1) b9(+1),b9(+1) a2(+1),a2(+1) 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) a9(+1),a9(+1) Mass (m/z) y15(+1),y % Intensity MALDI-PSD-MS b9(+1),b9(+1) b12(+1),b12(+1) b14(+1),b14(+1) y7(+1),y7(+1) 10 b10(+1),b10(+1) b6(+1),b6(+1) y2(+1),y2(+1) Mass (m/z) 5
6 100 Information from MS data : spot 2 PMF of a Bovine heart ventricle protein, Mr = 43 kda, pi = % Intensity Mass (m/z) Result : Creatine Kinase Information from MS-MS data MALDI-TOF/TOF MS of the Da peak b 2 b 4 b 8 LSVEALNSLTGEFK y y 12 y 10 y 8 y 6 y 4 70 b 4 % Intensity GGDDLDPNYVLSSR b b 4 b 3 -H 2 O y 11 y 10 y 8 y 5 y 6 y b b y 8 y y 10 y b8 y 9 y10 y y Mass (m/z) 6
7 Mass (m/z) MS-TAG results obtained with the MS/MS spectrum Information from MS data : spot 2 PMF of a Bovine heart ventricle protein, Mr = 43 kda, pi = % Intensity % Intensity m/_m = Mass (m/z) Result : Creatine Kinase 7
8 Mass (m/z) Information from MS data : spot 2 PMF of a Bovine heart ventricle protein, Mr = 43 kda, pi = m/_m = % Intensity % Intensity Mass (m/z) Result : Creatine Kinase ESI-MS/MS: the High-way for complex protein mixture identification 8
9 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
10 Peptide fragmentation during MS/MS sequencing From ESI ionisation source 10
11 Peptide fragmentation during MS/MS sequencing Peptide fragmentation during MALDI-MS/MS sequencing with MALDI ionisation source HGNTVLTALGGILKK b2(+1),b2(+1) b n ions % Intensity L,L,I,I 30 b4(+1),b4(+1),ntvl(+1) - 18,NTVL(+1) - 18 b3(+1),b3(+1) b5(+1),b5(+1) b4-18(+1),b4-18(+1) b6(+1),b6(+1) b8(+1),b8(+1) b12(+1),b12(+1) a2(+1),a2(+1) y10(+1),y10(+1) y2(+1),y2(+1) y7(+1),y7(+1) 10 y6(+1),y6(+1) b13(+1),b13(+1) b8-18(+1),b8-18(+1) y13(+1),y13(+1) Mass (m/z) 11
12 Peptide fragmentation during MS/MS sequencing From ESI ionisation source Database interrogation 12
13 Database interrogation Mascot search-output against SWISS-PROT database N-term side C-term side 13
14 Monocharged ions 28 Da 18 Da 18 Da 28 Da Peptide fragmentation during MS/MS sequencing 14
15 MS-Product from Protein prospector: Generate peptides theoretical fragments Peptide fragmentation during MS/MS sequencing From ESI ionisation source K A C -CAM C -CAM E E I/L T A E b 5 -H 2 O Y 10 -H 2 O Y 9 -H 2 O 15
16 Mascot search-output against SWISS-PROT database Internal fragment Peptide fragmentation during MS/MS sequencing From ESI ionisation source a 2 -H 2 O a 2 K A C -CAM C -CAM E E I/L T A E b 2 -H 2 O K Y b 2 x 3 b 5 -H 2 O Y 10 -H 2 O Y 9 -H 2 O 16
17 De novo bioinformatic tool approach De novo bioinformatic tool PEAKS: MS/MS de novo Peptide Sequencing ( /software/peaks/index.php) Real sequence : EYEATLEECCAK 17
18 Peptide fragmentation during MS/MS sequencing From ESI ionisation source De novo bioinformatic tool Real sequence : LVNELTEFAK 18
19 De novo bioinformatic tool De novo bioinformatic tool vs database search output LKECCDKPLLEK Mascot score: 39 (threshold: 35) 19
20 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
21 Bioinformatic tools for protein identification Database interrogation 21
22 MS/MS approach Mascot (public server or on-site server) MS-FIT (UCSF) ProFound (PROWL) 22
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