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
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
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, 30-40 m max salt) ften Automated w/ robotics high throughput (ALI-) ften good for visualizing Ps 3
Ion Chromatogram Peak chosen at 26.47 min Ion Current over 60 min ass at 571.36 chosen for S/S S/S S 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 [+] 4
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
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 6
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 7
SRI-CI ASELAEVR + + msec to sec, collision gas in ICR Is this a good (informative) fragmentation spectrum?? y 14 y 5 y 9 y 11 15 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
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
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 71.09 Arginine AR R 156.19 artic Acid ASP 115.1 aragine AS 114.09 Cysteine CYS C 103.15 lutamic Acid LU E 129.12 lutamine L Q 128.14 lycine LY 57.05 istidine IS 137.14 Isoleucine ILE I 113.16 Leucine LEU L 113.16 Lysine LYS K 128.17 hionine E 131.19 nylalanine PE 147.18 Proline PR P 97.12 Serine SER S 87.08 eonine R 101.11 ryptophan RP W 186.12 yrosine YR Y 163.18 Valine VAL V 99.14 Internal cleavage ions immonium ions http://www.matrixscience.com/help/fragmentation_help.html 10
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 11
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? 12
An S/S spectrum of the m/z = 571.4 peptide. We will sequence this together 13
14
ly ly 15
ly ly 16
ly ly 17
Build the peptide: selected peptide = 1141.4 Estimate the number of amino acids ly Possibly 10 amino acids Consider a y-ion series ly 18
1141 1141.4 selected + ly 1141 1141.4 selected +.6 Largest fragment observed ly 19
1141 1141.4 selected +.6 Largest fragment observed 98.8 difference Is there an amino acid with that mass? ly V 1141 99 = Valine he missing amino acid What is the next mass observed? ly 20
V ly V ly 21
V ly V ly 22
V ly V ly 23
V ly V ly 24
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
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
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 262 262 = smallest identified fragment - 19 = mass of + + 243 = mass of missing amino acids What amino acids? ly 27
V 262 int: ryptic! 262 = smallest identified fragment - 19 = mass of + + 243 = mass of missing amino acids What amino acids? ly V 262 262 87 = Serine 156 = Arginine 243 19 = mass of + + 115 = artic Acid 128 = Lysine 243 19 = mass of + + 262 ly 28
200 400 600 800 1000 1200 1400 1600 m/z 120000 100000 80000 60000 40000 20000 0 200 400 600 800 1000 1200 1400 1600 m/z 7/23/2014 V S R 262 87 = Serine 156 = Arginine 243 19 = mass of + + 262 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
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 V S R 262 87 = Serine 156 = Arginine 243 19 = mass of + + 262 ly 30
his works because the fragments are predictable VSR Experimental 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 31
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 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
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