1 / 39

Manual De Novo Peptide MS/MS Interpretation For Evaluating Database Search Results Karl R. Clauser

Understand AA properties, fragmentation pathways, ion types, charge dynamics, and more for effective MS/MS interpretation of peptide sequences in proteomics research. Practical guidelines for handling sample artifacts and ambiguities.

ccalkins
Download Presentation

Manual De Novo Peptide MS/MS Interpretation For Evaluating Database Search Results Karl R. Clauser

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Manual De Novo Peptide MS/MS Interpretation For Evaluating Database Search Results Karl R. Clauser Broad Institute of MIT and Harvard Cold Spring Harbor Proteomics Course July, 2010

  2. Outline • AA properties • Fragmentation pathways and ion types • b/y pairs • Fragment charge from mass defect • Non-mobile proton • Neutral loss ion types • Phosphosite ambiguity • Sample handling chemistry artifacts • Isobaric co-eluters • Mass tolerance units and isobaric AA’s • Other Tutorials • Dominant ions • AA adjacencies • Positions

  3. AA Structures & Masses pK: N-term 7.5 pK: C-term 3.5 pK: 10 6 12 pK: 4.0 4.5 D E 115 129 S T Y 87 101 163 N Q 114 128 P 97 L I 113 113 M C 131 103 (+57 IAA) F W 147 186 G A V 57 71 99 K  H R 128 137 156 Name AA Mass Gly G 57 Ala A 71 Ser S 87 Val V 99 Thr T 101 Leu/Ile L/I 113 Asn N 114 Asp D 115 Lys/Gln K/Q 128 Glu E 129 Met M 131 His H 137 Phe/Met-ox F/m 147 Arg R 156 Cys-IAA C 160 Tyr Y 163 Trp W 186 http://ionsource.com/Card/clipart/aaclipart.htm

  4. R1 R2 R3 R4 O O O O + H2N CH C NH CH C NH CH C NH CH C OH H R3 R1 R2 H R4 Charge-directed Fragmentation Scheme zHz+ O O O O + H2N CH C NH CH C NH CH C NH CH C OH H b ion formation y ion formation and/or y1 b3 + + Neutral pumped away by vacuum system + Neutral pumped away by vacuum system Proton Mobility Mobile: zpre > #Arg + #Lys + #His Partially mobile: zpre< #Arg + #Lys + #His and > #Arg Non-mobile: zpre< #Arg For peptides with non-mobile protons, fragmentation tends to proceed via charge-remote mechanisms. MS/MS spectra will be dominated by a few ions, typically: C-term side of D, E N-term side of P

  5. O O O O H2N CH C NH CH C NH CH C NH CH C OH R1 R2 R3 R4 Sequence Specfic Fragment Ion Types a3 b3 c3 nHn+ x1 y1 z1 Ion type restrictions residues delta a-NH3 contains NH3 residue RK NQ -17 b-NH3, y-NH3 contains NH3 residue RK NQ -17 b-H2O, y-H2O contains H2O residue ST DE -18 b-H3PO4, y-H3PO4 contains H3PO4 residue st -98 y++, b++ contains charged residues RHK

  6. 128 99 99 128 E VQ L V|E/S|G|G|GL|V|K|PG G\S\L\R Complementary Ions b/y pairs

  7. Dual Picket Fence 163 163 11371101115 115 101 71 113 163 163 A E/D|T|A|L|Y|Y|CA\K

  8. Uniqueness of a Peptide Sequence Clauser, K. R.; Baker, P. R.; Burlingame, A. L. " Role of Accurate Mass Measurement ( +/- 10ppm) in Protein Identification Strategies Employing MS or MS/MS and Database Searching", Anal. Chem. 1999, 71, 2871-2882.

  9. Diagnose Doubly Charged Fragment Ions I/A|D|A|H|L|D|R

  10. Dominant Cleavage Proline N-side 28 87 97 b2 N F|P/S/P V DA A F R y9

  11. 202 115 115 202 (K)I S R|P G D|SD|D|SR(S) Non-mobile proton zpre< #Arg Sparse Dominant Fragmentation

  12. Cry Babies (b-H2O & b pairs) P(m/z)-2H2O P(m/z)-H2O E/H/A|V/E|G/D|C D|F Q L L K

  13. Andrea Kaitlin Aidan Jack Interpreting MS/MS Spectra is Fun!!

  14. Andrea Kaitlin Aidan Jack Interpreting MS/MS Spectra is Fun!!

  15. Source of Incorrect MS/MS Interpretations • Major • Database • Peptide not in database. Mutation. MS/MS not from a peptide. • Unanticipated Protein Chemistry • Chemical modification, post-translational modification. • Enzyme/Ion Source • Non-specific cleavage. In-source fragmentation yields MS3. • Minor • Algorithm • Fragment ion types of instrument not accounted for. Peak Detection. • Instrument Resolution • Wrong parent charge. Wrong fragment charge. • User Competence • Wrong parameters selected.

  16. Phospho Site Ambiguity – S/T P(m/z)-H3PO4-H2O P(m/z)-H3PO4 P(m/z)-H3PO4-2H2O L P/S s/P/V|Y/E/D|A A S F K

  17. Phospho Site Ambiguity – S/T L A G G Q/T/S Q|P T T|P L\T s/P Q R L A G G Q/T/S Q|P T T|P L\t S/P Q R

  18. “Resulting sequences were inspected manually …. When the exact site of phosphorylation could not be assigned for a given phosphopeptide, it was tabulated as ambiguous.” “All spectra supporting the final list of assigned peptides used to build the tables shown here were reviewed by at least three people to establish their credibility.” “Assignment of phosphorylation sites was verified manually with the aid of PEAK Studio (Bioinformatics Solutions) software.” “All identified phosphopeptides were manually validated, and localization of phosphorylated residues within the individual peptide sequences were manually assigned…” Reliability of LC/MS/MS Phosphoproteomic Literature Citation Approach Instrument #sites #ambiguous Scores Site Supplem. sites Shown Ambiq Labeled Shown Spectra Ballif, BA,…Gygi, SP 1DGel LCQ Deca XP 546 86 yes yes no 2004 MCP, 3, digest, SCX 1093-1101 LC/MS/MS Rush, J, … Comb, MJ digest lysate LCQ Deca XP 628 0 yes no no 2005, Nat Biotech, 23, pTyr Ab 94-101 LC/MS/MS Collins, MO, …Grant, SGN protein IMAC Q-Tof Ultima 331 42 no yes no 2005, J Biol Chem, 280, peptide IMAC 5972-5982 LC/MS/MS Gruhler, A, … Jensen, ON digest lysate LTQ-FT 729 0 yes no no 2005 MCP, 4, SCX, IMAC 310-327 LC/MS/MS

  19. Expect Woes & Nuisances • Sample Handling Chemistry • Carbamylation +43 nterm, Lys urea in digest buffer • Deamidation +1 N -> D sample in acid • pyroGlutamic acid -17 nterm Q sample in acid • Oxidized Met +16 M gels • Cys alkylation reagent +x n-term, W • Data Dependent Acquisition Parameters • Isobaric Co-eluters • Protein Isoforms / Family Members • Isobaric peptides from related proteins

  20. Stinkers (b-NH3) & Pyroglutamic Acid (R)q L/Q|L|A|Q|E|A|A\Q\K(R) -17 Da Q to q (R)Q L/Q/L/A|Q/E/A|A Q\K(R) P(m/z)-NH3

  21. G S/E/S|G|I|F|T|n\T K 18.35 96.9% +0.007 Da G S/E/S|G|I|F|T|D\T K Deamidation G S/E S\G\I\F\T\N/T K 6.62 43.4% +0.986 Da

  22. Deamidation of Asn +1Da Asn –NH + O = Asp ionsource.com

  23. Carbamylation from Urea in Digest Buffer +43Da CNHO +43Da

  24. Carbamylated N-term I/G/E|G/T/y/G V|V|Y\K P(m/z)-CNHO +43 b ions P(m/z)-CNHO-H2O

  25. Merged 4 spectra same precursor 50 sec window different peptides Know Your Chromatographic Peak Widths (K)E E m E S A E G|L|K\G P/m\K(S) Top Database Search Result 8.78 71.0% DFwdRev: 3.49

  26. (R)R G G/P P\F A\F|V|E|F|E|D|P R(D) (R)N P P R\F A\F|V|E|F|E|D|P\R(D) Related Proteins : Distinct Non-differentiable Peptides

  27. >0.8 0.4 - 0.8 # dominant ions # total cleavages 0.1 - 0.4 - (<3 obsv) Frequency of Dominance at Adjacent AA’s – v9, z=2 Mobile Partially Mobile 2061 spectra 4525 spectra Non-mobile 114 spectra

  28. 67% 72% 76% Frequency and Distribution Dominant Ions v9 5758 2974 177 Proton Mobility Mobile: zpre > #Arg + #Lys + #His Partially mobile: zpre< #Arg + #Lys + #His and > #Arg Non-mobile: zpre< #Arg Precursor z=2, 6699 spectra from a trypsin GeLC/MS/MS experiment on an LTQ-FT

  29. y++-h2o @ yn-2 E at position 3 (K)V/A|E|I/E|H|A\E\K(E) Position-dependent Dominant Ions

  30. Short Peptides Often Yield a Dominant Ion Cleavage Between Residues 2 & 3 Bonus C-side b2 residues at position 3: PRKH Bonus N-side b2 residues at position 1 or 2: PRKHNQqVILFYW Bonus ignore b2: niether of above but still dominant If there is a mobile or partially mobile proton, peptides of length <14 are likely to yield at least one intense fragment ion between residues 2 and 3 (yellow and pink curves shifted to shorter lengths, purple curve shifted to longer lengths). Intense ions are favored by the presence of PRKH at residue 3 or the presence of PRKHNQqVILFYW at residues 1 or 2.

  31. Dominant Ions – Mobile b2/yn-2 v25 b2/yn-2 (K)A N|S/N/L/V L|Q|A|D\R(S)

  32. Physiochemical Complications to Computational Interpretation • Incomplete Fragmentation • Inconsistent intensity of fragment ion types • Instrument type dependent • Amino acid dependent • Isobaric AA’s • I = L (C6 H11 N1 O) • K = Q (C6 H12 N2 O, C5 H8 N2 O2) • Isobaric AA combinations • GG=N (C4 H6 N2 O2 , C4 H6 N2 O2) • GA=K=Q (C5 H8 N2 O2, C6 H12 N2 O, C5 H8 N2 O2) • W=DA=VS (C11 H11 N2 O, C7 H10 N2 O4, C8 H14 N2 O3) • Parent charge uncertainty • Fragment charge uncertainty • Chemical or post-translational modifications

  33. Consequences of Inappropriate Tolerance Units (using Da tolerance when instrument errors are in ppm) too loose too tight just right • Isobaric AA’s • I = L (C6 H11 N1 O) = 113.08406 • K ~ Q (C6 H12 N2 O, C5 H8 N2 O2) 128.09496 ~ 128.05858 D =0.03638 • F~m (C9 H9 N O, C5 H9 N O S) 147.06841 ~ 147.0354 D =0.0330 • Isobaric AA combinations • GG=N (C4 H6 N2 O2 , C4 H6 N2 O2) 114.04293 • GA=Q~K (C5 H8 N2 O2, C5 H8 N2 O2, C6 H12 N2 O) 128.09496 ~ 128.05858 D =0.03638 • DA~W~VS (C7 H10 N2 O4, C11 H11 N2 O, C8 H14 N2 O3) 186.06405 ~ 186.07931 ~ 186.10044 D =0.01526 D =0.02113

  34. Additional Resources Google: “de novo sequencing tutorial” Don Hunt and Jeff Shabanowitz - manual http://www.ionsource.com/tutorial/DeNovo/DeNovoTOC.htm Rich Johnson - manual http://www.abrf.org/ResearchGroups/MassSpectrometry/EPosters/ms97quiz/SequencingTutorial.html PEAKS - automated http://www.bioinformaticssolutions.com/products/peaks/support/tutorials/PEAKS_De_Novo.html

  35. Acknowledgements Broad Institute Steve Carr Terri Addona Jinyan Du Phillip Mertins MIT Michael Yaffe Majbrit Hjerrld Drew Lowery

  36. Near Self Reversal

  37. 10.37 (K) L/G|F/S/L t/P/S K (G) (K) L/G|F/s/L T/P/S K (G) 10.60

More Related