MS/MS of Peptides Manual Sequencing of Protonated Peptides

Transcription

1 S/S of Peptides anual Sequencing of Protonated Peptides Árpád Somogyi Associate irector CCIC, ass Spectrometry and Proteomics Laboratory SU July 11, 2018 Peptides Product Ion Scan Product ion spectra contain many types of fragment ions charge directed charge remote a, b, y type sequencing ions internal fragments, immonium ions Important for sequencing amino acid determined from mass between peaks in spectrum y ions series b ions series immonium ions (identify amino acids in the peptide) a ions (confirm b ion after a loss of C, 28 amu) Presented here: peptide fragment ions a mechanism for fragment ion formation a peptide to sequence 1

2 A mechanism of peptide fragmentation (2) ucleophilic attack (Peptide) C C (1) positive charge R 2 C C C C (Peptide) R 1 R 3 (Peptide) C R 1 R 2 C C C 3 (3) cyclic intermediate (Peptide) Ref: Wysocki, 2000 (Peptide) C R 1 A mechanism of peptide fragmentation R 2 C C (4) Rearrangement C 3 (Peptide) (Peptide) C R 1 R 2 C 3 C C (Peptide) 2 (Peptide) C R 1 R C C R 3 (Peptide) b oxazolone ion neutral Ref: Wysocki,

3 A mechanism of peptide fragmentation (Peptide) C R 1 R 2 C C (Peptide) (4) Rearrangement C 3 (Peptide) C R 1 R 2 C C 2 (Peptide) C 3 (Peptide) C R 1 R C C R 3 (Peptide) oxazolone neutral (or other structure) y ion Ref: Wysocki, 2000 Acidic group of () can cause cleavage Ref: Wysocki,

4 Peptides fragment in a predictable way 2 + -terminal = a/b ions + neutral or + C-terminal = neutral + y ion b 2 b 3 2 R 1 a 2 a 3 If doubly charged R 3 parent: possible b/y ion pair or doubly charged b or y fragment ions R 2 R 4 R 5 y 3 y 2 Peptides fragment in a predictable way resulting in a series of peptide fragment ions a 2 b 2 c 2 Peptide bond fragment ions 2 C C C C C C C C z 2 2 R C C C x 2 y 2 R R' 2 C Internal immonium ion Amino acid immonium ion 4

5 b/y ion series commonly used for sequencing common with CI Alternative activation methods (E, EC) generate c/z ion series Can also be used to sequence peptides 1 c/z Ion formation mechanism Chemot-Rooke et al. J Am Soc ass Spectrom 2007, 18,

6 2 c/z Ion formation mechanism Chemot-Rooke et al. J Am Soc ass Spectrom 2007, 18, between ion series = Residue mass Peptide bond fragment ions b 1 b 2 b 3 2 C C C C C C C C y 3 m/z 190 y 2 m/z 133 y 1 m/z 76 Residue ass specific to amino acid present in sequence 6

7 Peptide Sequencing C 71 u. 115 u. Ala C C C 3 C C C 2 C amino acid abbreviations residue mass Alanine ALA A 71.1 Arginine AR R aragine AS artic Acid ASP Cystein CYS C lutamic Acid LU E lutamine L Q cine LY 57.1 istidine IS Isoleucine ILE I Leucine LEU L Lysine LYS K hionine E nylalanine PE Proline PR P 97.1 Serine SER S 87.1 hreonine R ryptophan RP W yrosine YR Y Valine VAL V 99.1 Internal cleavage ions immonium ions 7

8 LEARI CECK ryptic Peptide Sequencing Exercise performed in a low res ion trap S/S Ion Chromatogram Peak chosen at min Ion Current over 60 min ass at chosen for S/S S/S S 8

9 Peptide precursor ions observed by S [+ 2] 2+ m/z = m/z = calculation of m/z measured x 2 1,142.4 [+2] ,141.4 [+] S/S of his spectrum will tell us peptide sequence eventually W? 9

10 An S/S spectrum of the m/z = peptide. We will sequence this together 10

11 11

12 hr 12

13 hr hr 13

14 hr hr 14

15 Build the peptide: selected peptide = Estimate the number of amino acids hr Possibly 10 amino acids Consider a y ion series hr 15

16 selected + hr selected +.6 Largest fragment observed hr 16

17 selected +.6 Largest fragment observed 98.8 difference Is there an amino acid with that mass? hr V = Valine he missing amino acid What is the next mass observed? hr 17

18 V hr V hr 18

19 V hr V hr 19

20 V hr V hr 20

21 V hr V hr 21

22 V 262 If this is a y ion series: 262 = smallest ion in the series what does it represent? hr V 262 All amino acids in table are peptide bond to peptide bond 71 u. 115 u. Ala hr C C C C C C 3 C 2 C 22

23 V 262 We re missing one terminal hydrogen 71 u. 115 u. Ala hr C C C C C C 3 C 2 C V 262 We re missing one C terminal roup 71 u. 115 u. Ala hr C C C C C C 3 C 2 C 23

24 V 262 And the ionizing proton otal = 19 amu 71 u. 115 u. Ala hr + C C C C C C 3 C 2 C V = smallest identified fragment 19 = mass of = mass of missing amino acids What amino acids? hr 24

25 V 262 int: ryptic! 262 = smallest identified fragment 19 = mass of = mass of missing amino acids What amino acids? hr V = Serine 156 = Arginine = mass of = artic Acid 128 = Lysine = mass of hr 25

26 V S R = Serine 156 = Arginine = mass of hr V S R = Serine 156 = Arginine = mass of hr 26

27 Sequencing a Peptide Peptide ass +(monoisotopic): y y2 99 V y3 129 E y4 71 A y5 71 A y6 Sequence ELAAEVR 113 L y7 129 E y b b b b b b b m/z Computer programs search databases that contain information and sequence of proteins gi sp P07740 LUXA_VIBA Alkanal monooxygenase alpha chain (Bacterial luciferase alpha chain) KLLYQPPELSQEVKRLVLKASECVWLLEELL PYVAAALLAELVAAIVLPAPVRQAEVLLQSKRRICR LYKRVSRALCWYLKEEYIAAEIKPKIQLP SAYQAPVYVVAESASEWAAERLPILSWIIEKKAQLLYEVAE YVKICLSYISVSRAKICRLWYSYVAKISQK YKQWRVLKKRRIYSYEIPVPEECIAIIQQIAIICC EASEEEIIASKLQSVPYLKEKQ = K = LLYQPPELSQEVK = R = LVLK = ASECVWLLEELLP YVAAALLAELVAAIVLPAPVR = QAEVLLQSK = R = R easured peptide = = ICR = LYK = R = VSR 27

28 his works because the fragments are predictable Experimental VSR heoretical Protein Prospector: mass intensity VSR ore peptides identified increases confidence in I ISQK LVLK QAEVLLQSK If all of these peptides belonged to an unknown protein, S/S could potentially reveal protein identity 28

29 gi sp P07740 LUXA_VIBA Alkanal monooxygenase alpha chain (Bacterial luciferase alpha chain) KLLYQPPELSQEVKRLVLKASECVWLLEE LLPYVAAALLAELVAAIVLPAPVRQAEVLLQSK RRICRLYKRVSRALCWYLKEEYIAA EIKPKIQLPSAYQAPVYVVAESASEWAAERLPILSWIIE KKAQLLYEVAEYVKICLSYISVSRAKICRLWY SYVAKISQKYKQWRVLKKRRIYSYEIPV PEECIAIIQQIAIICCEASEEEIIASKLQSVPYLKEK Q Sequence more peptides VSR emonstration Sequence ISQK Spectra 1 VYLEEVR Spectra 2 ESYSEQK Spectra 3 ISQK Spectra 4 LYKR Spectra 5 ote that Peptide 1 & 4 are the same 1 = doubly charged precursor 4 = singly charged precursor 29