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
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 2 + 2 C C R 3 (Peptide) b oxazolone ion neutral Ref: Wysocki, 2000 2
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 2 + 2 C C R 3 (Peptide) oxazolone neutral (or other structure) y ion Ref: Wysocki, 2000 Acidic group of () can cause cleavage Ref: Wysocki, 2000 3
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
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, 2146-2161 5
2 c/z Ion formation mechanism Chemot-Rooke et al. J Am Soc ass Spectrom 2007, 18, 2146-2161 between ion series = Residue mass Peptide bond fragment ions b 1 b 2 b 3 2 C C C C C C C C 57 57 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
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 156.2 aragine AS 114.1 artic Acid ASP 115.1 Cystein CYS C 103.2 lutamic Acid LU E 129.1 lutamine L Q 128.1 cine LY 57.1 istidine IS 137.1 Isoleucine ILE I 113.2 Leucine LEU L 113.2 Lysine LYS K 128.2 hionine E 131.2 nylalanine PE 147.2 Proline PR P 97.1 Serine SER S 87.1 hreonine R 101.1 ryptophan RP W 186.1 yrosine YR Y 163.2 Valine VAL V 99.1 Internal cleavage ions immonium ions http://www.matrixscience.com/help/fragmentation_help.html 7
LEARI CECK ryptic Peptide Sequencing Exercise performed in a low res ion trap S/S Ion Chromatogram Peak chosen at 26.47 min Ion Current over 60 min ass at 571.36 chosen for S/S S/S S 8
Peptide precursor ions observed by S [+ 2] 2+ m/z = 571.2 + m/z = 1141.3 calculation of + 571.2 m/z measured x 2 1,142.4 [+2] - 1.0 1,141.4 [+] S/S of 571.2 his spectrum will tell us peptide sequence eventually W? 9
An S/S spectrum of the m/z = 571.4 peptide. We will sequence this together 10
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hr 12
hr hr 13
hr hr 14
Build the peptide: selected peptide = 1141.4 Estimate the number of amino acids hr Possibly 10 amino acids Consider a y ion series hr 15
1141 1141.4 selected + hr 1141 1141.4 selected +.6 Largest fragment observed hr 16
1141 1141.4 selected +.6 Largest fragment observed 98.8 difference Is there an amino acid with that mass? hr V 1141 99 = Valine he missing amino acid What is the next mass observed? hr 17
V hr V hr 18
V hr V hr 19
V hr V hr 20
V hr V hr 21
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
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
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 262 262 = smallest identified fragment 19 = mass of + + 243 = mass of missing amino acids What amino acids? hr 24
V 262 int: ryptic! 262 = smallest identified fragment 19 = mass of + + 243 = mass of missing amino acids What amino acids? hr V 262 262 87 = Serine 156 = Arginine 243 19 = mass of + + 115 = artic Acid 128 = Lysine 243 19 = mass of + + 262 hr 25
V S R 262 87 = Serine 156 = Arginine 243 19 = mass of + + 262 hr V S R 262 87 = Serine 156 = Arginine 243 19 = mass of + + 262 hr 26
Sequencing a Peptide Peptide ass +(monoisotopic): 1025.5374 175.23 y1 137 312.31 y2 99 V 411.36 y3 129 E 540.38 y4 71 A 611.43 y5 71 A 682.42 y6 Sequence ELAAEVR 113 L 795.46 y7 129 E 924.58 y8 231.18 b2 344.23 b3 415.26 b4 486.34 b5 615.28 b6 714.37 b7 851.35 b8 200 300 400 500 600 700 800 900 m/z Computer programs search databases that contain information and sequence of proteins gi 126509 sp P07740 LUXA_VIBA Alkanal monooxygenase alpha chain (Bacterial luciferase alpha chain) KLLYQPPELSQEVKRLVLKASECVWLLEELL PYVAAALLAELVAAIVLPAPVRQAEVLLQSKRRICR LYKRVSRALCWYLKEEYIAAEIKPKIQLP SAYQAPVYVVAESASEWAAERLPILSWIIEKKAQLLYEVAE YVKICLSYISVSRAKICRLWYSYVAKISQK YKQWRVLKKRRIYSYEIPVPEECIAIIQQIAIICC EASEEEIIASKLQSVPYLKEKQ 278.39 = K 2343.72 = LLYQPPELSQEVK 175.21 = R 643.80 = LVLK 5887.73 = ASECVWLLEELLP YVAAALLAELVAAIVLPAPVR 1491.67 = QAEVLLQSK 232.26 = R 322.38 = R easured peptide = 1141.4 595.74 = ICR 595.67 = LYK 437.47 = R 1142.23 = VSR 27
his works because the fragments are predictable Experimental VSR heoretical Protein Prospector: http://prospector.ucsf.edu/ mass intensity 218.9 154041 219.0 185821 246.9 617130 376.2 262276 377.1 132554 387.0 121560 439.2 307984 439.3 78476 491.1 214504 503.3 111570 512.7 269518 512.8 347071 520.0 209338 521.5 133052 521.9 308869 522.2 70668 562.2 101171 562.5 114861 622.2 521627 623.2 80720 633.1 179525 737.2 494965 738.2 106430 738.3 202034 766.2 88816 838.2 201007 877.2 262518 878.2 73913 895.2 2305621 896.2 480302 896.3 1027102 897.3 260690 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
gi 126509 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