Introduction to Proteomics: Fragmentation of protonated peptides and manual sequencing
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1 Introduction to Proteomics: ragmentation of protonated peptides and manual sequencing Árpád Somogyi CCIC SP SU Summer Workshop S/S hod using ESI Ion rap (Bottom up) 1
2 An alternative strategy for complex mixtures UPI LC/LC S/S SCX = Strong cation exchange RP = Reverse Phase (C 18) Alternate an increasing salt gradient (move some peptides onto RP) ollow by RP gradient (separate peptides, send to mass spec) SCX RP S/S peptides from many proteins Results in thousands of mass spectra A computational challenge! udpi In chromatographic theory, theoretical plates of orthogonal separation columns back-to-back are multiplied rather than summed that s why it works Load peptide mixture SCX Salt Bump SCX RP RP o S o S Reverse Phase radient SCX RP o S 2
3 ypes of Separation echnologies for olecules hod el Electrophoresis (1) el Electrophoresis (2) Reverse Phase (C8 or C4) chromatography Separation based on olecular mass Isoelectric point (pi; IE) & olecular mass Combination of hydrophobicity and molecular weight Separation done using el (which acts like a molecular sieve) and potential el, potential and ampholytes PLC urther steps In-gel digestion of proteins to peptides LC- S/S or ALI- -S el iltration olecular Weight PLC Ion Exchange Cation or Anion affinity PLC Protein(s) igest to peptides LC- S/S or ALI- -S Affinity Chromatography A,RA, Anti-body, peptides etc PLC ud-pi (ultidimensional Protein Identification echnology Cation Exchange & hydrophobicity (used for peptides; not for proteins) PLC nline S/S analysis 2 Electrophoresis Protein ixture or IP eluant or Cell/tissue 2 SS-PAE reat clean-up tool (rid of salts, detergents, etc ) Various stains available various detection limits Protein profiling Various p ranges 2 gels are very much sample related (sample may require further clean-up prior to 2 gel, e.g., dialysis, desalting, etc.) Avoid excess salts in sample (not focus, IPs burn, m max salt) ften Automated w/ robotics high throughput (ALI-) ften good for visualizing Ps 3
4 Ion Chromatogram Peak chosen at min Ion Current over 60 min ass at chosen for S/S S/S S 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 [+] 4
5 Result is many S/S spectra ow do we determine the aa sequence? Peptides Product Ion Scan (obile Proton odel) 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 5
6 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,
7 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,
8 SRI-CI ASELAEVR + + msec to sec, collision gas in ICR Is this a good (informative) fragmentation spectrum?? y 14 y 5 y 9 y 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 8
9 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 Peptides fragment in a predictable way 2 + -terminal = a/b ions + neutral or + C-terminal = neutral + y ion b 2 b 3 oubly charged parent? possible b/y ion pair a 2 a 3 R 1 R 3 R 5 2 R 2 R 4 y 3 y 2 9
10 asses of amino acids between peptide bonds C 71 u. 115 u. Ala C C C 3 C C C 2 C amino acid mass Alanine ALA A Arginine AR R artic Acid ASP aragine AS Cysteine CYS C lutamic Acid LU E lutamine L Q lycine LY istidine IS Isoleucine ILE I Leucine LEU L Lysine LYS K hionine E nylalanine PE Proline PR P Serine SER S eonine R ryptophan RP W yrosine YR Y Valine VAL V Internal cleavage ions immonium ions 10
11 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 11
12 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? 12
13 An S/S spectrum of the m/z = peptide. We will sequence this together 13
14 14
15 ly ly 15
16 ly ly 16
17 ly ly 17
18 Build the peptide: selected peptide = Estimate the number of amino acids ly Possibly 10 amino acids Consider a y-ion series ly 18
19 selected + ly selected +.6 Largest fragment observed ly 19
20 selected +.6 Largest fragment observed 98.8 difference Is there an amino acid with that mass? ly V = Valine he missing amino acid What is the next mass observed? ly 20
21 V ly V ly 21
22 V ly V ly 22
23 V ly V ly 23
24 V ly V ly 24
25 V 262 If this is a y-ion series: 262 = smallest ion in the series what does it represent? ly V 262 All amino acids in table are peptide bond to peptide bond 71 u. 115 u. Ala ly C C C C C C 3 C 2 C 25
26 V 262 We re missing one -terminal hydrogen 71 u. 115 u. Ala ly C C C C C C 3 C 2 C V 262 We re missing one C-terminal roup 71 u. 115 u. Ala ly C C C C C C 3 C 2 C 26
27 V 262 And the ionizing proton otal = 19 amu 71 u. 115 u. Ala ly + 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? ly 27
28 V 262 int: ryptic! 262 = smallest identified fragment - 19 = mass of = mass of missing amino acids What amino acids? ly V = Serine 156 = Arginine = mass of = artic Acid 128 = Lysine = mass of ly 28
29 m/z m/z 7/23/2014 V S R = Serine 156 = Arginine = mass of ly ata cannot be manually analyzed, instead, computer programs do it S/S? protein peptides identify rank S Peptide W ound in Selected atabases ALYP... SWLAL... PLVSY... Relative Intensity theoretical spectra compare Relative Intensity 29
30 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 V S R = Serine 156 = Arginine = mass of ly 30
31 his works because the fragments are predictable VSR Experimental 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 31
32 gi sp P07740 LUXA_VIBA Alkanal monooxygenase alpha chain (Bacterial luciferase alpha chain) KLLYQPPELSQEVKRLVLKASECVWLLEE LLPYVAAALLAELVAAIVLPAPVRQAEVLLQSK RRICRLYKRVSRALCWYLKEEYIAA EIKPKIQLPSAYQAPVYVVAESASEWAAERLPILSWIIE KKAQLLYEVAEYVKICLSYISVSRAKICRLWY SYVAKISQKYKQWRVLKKRRIYSYEIPV PEECIAIIQQIAIICCEASEEEIIASKLQSVPYLKEK Q Sequence more peptides 5 S/S spectra are located in the back of this section Identify the peptide in each Actually, identify as many amino acids as you can We ll give you some time, then show the sequences 32
33 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 33
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