1. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 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. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 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. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 3 AA Structures & Masses http://ionsource.com/Card/clipart/aaclipart.htm G A V 57 71 99 S T Y 87 101 163 F W 147 186 P 97 M C 131 103 (+57 IAA) L I 113 D E 115 129 pK: 4.0 4.5 pK: C-term 3.5 K H R 128 137 156 N Q 114 128 pK: 10 6 12 pK: N-term 7.5 NameAA Mass GlyG57 AlaA71 SerS87 ValV99 ThrT101 Leu/IleL/I113 AsnN114 AspD115 Lys/GlnK/Q128 GluE129 MetM131 HisH137 Phe/Met-oxF/m147 ArgR156 Cys-IAAC160 TyrY163 TrpW186

4. Karl Clauser Proteomics and Biomarker Discovery H 2 N CH C NH CH C NH CH C NH CH C OH R1R1 R2R2 R3R3 R4R4 OOOO H b3b3 b ion formation NH CH C OH R4R4 O H + H y1y1 y ion formation Charge-directed Fragmentation Scheme + Neutral pumped away by vacuum system and/or H 2 N CH C NH CH C NH CH C R1R1 R2R2 OOO R3R3 zH z+ + + Neutral pumped away by vacuum system + Proton Mobility Mobile:z pre > #Arg + #Lys + #His Partially mobile:z pre #Arg Non-mobile:z pre < #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. Karl Clauser Proteomics and Biomarker Discovery H 2 N CH C NH CH C NH CH C NH CH C OH R1R1 R2R2 R3R3 R4R4 OOOO y1y1 b3b3 nH n+ Sequence Specfic Fragment Ion Types z1z1 x1x1 a3a3 c3c3 Ion type restrictionsresiduesdelta a-NH 3 contains NH 3 residueRK NQ-17 b-NH 3, y-NH 3 contains NH 3 residueRK NQ-17 b-H 2 O, y-H 2 Ocontains H 2 O residue ST DE-18 b-H 3 PO 4, y-H 3 PO 4 contains H 3 PO 4 residuest -98 y++, b++contains charged residues RHK

6. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 6 Complementary Ions b/y pairs E V Q L V|E/S|G|G|G L|V|K|P G G\S\L\R 128 99 99 128

7. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 7 Dual Picket Fence A E/D|T|A|L|Y|Y|C A\K 115 101 71 113 163 163 163 163 113 71 101 115

8. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 8 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. Uniqueness of a Peptide Sequence

9. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 9 I/A|D|A|H|L|D|RI/A|D|A|H|L|D|R Diagnose Doubly Charged Fragment Ions

10. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 10 Dominant Cleavage Proline N-side N F|P/S/P V D A A F R y9y9 b2b2 2887 97

11. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 11 Sparse Dominant Fragmentation 115 202 202 115 (K)I S R|P G D|S D|D|S R(S) Non-mobile proton z pre < #Arg

12. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 12 E/H/A|V/E|G/D|C D|F Q L L K Cry Babies (b-H 2 O & b pairs) P(m/z)-H 2 O P(m/z)-2H 2 O

13. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 13 Interpreting MS/MS Spectra is Fun!! Kaitlin AidanJack Andrea

14. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 14 Interpreting MS/MS Spectra is Fun!! Kaitlin AidanJack Andrea

15. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 15 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 MS 3. 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. Source of Incorrect MS/MS Interpretations

16. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 16 Phospho Site Ambiguity – S/T L P/S s/P/V|Y/E/D|A A S F K P(m/z)-H 3 PO 4 -H 2 O P(m/z)-H 3 PO 4 P(m/z)-H 3 PO 4 -2H 2 O

17. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 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. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 18 CitationApproachInstrument#sites#ambiguousScoresSiteSupplem. sitesShownAmbiqLabeled Shown Spectra Ballif, BA,…Gygi, SP1DGelLCQ Deca XP54686yesyesno 2004 MCP, 3,digest, SCX 1093-1101LC/MS/MS Rush, J, … Comb, MJdigest lysate LCQ Deca XP6280yesnono 2005, Nat Biotech, 23,pTyr Ab 94-101LC/MS/MS Collins, MO, …Grant, SGNprotein IMACQ-Tof Ultima33142noyesno 2005, J Biol Chem, 280,peptide IMAC 5972-5982 LC/MS/MS Gruhler, A, … Jensen, ONdigest lysateLTQ-FT7290yesnono 2005 MCP, 4,SCX, IMAC 310-327LC/MS/MS Reliability of LC/MS/MS Phosphoproteomic Literature “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 identified phosphopeptides were manually validated, and localization of phosphorylated residues within the individual peptide sequences were manually assigned…” “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.”

19. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 19 Sample Handling Chemistry Carbamylation+43nterm, Lysurea in digest buffer Deamidation +1 N -> Dsample in acid pyroGlutamic acid-17nterm Qsample in acid Oxidized Met+16Mgels Cys alkylation reagent +xn-term, W Data Dependent Acquisition Parameters Isobaric Co-eluters Protein Isoforms / Family Members Isobaric peptides from related proteins Expect Woes & Nuisances

20. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 20 (R)q L/Q|L|A|Q|E|A|A\Q\K(R) (R)Q L/Q/L/A|Q/E/A|A Q\K(R) P(m/z)-NH 3 Stinkers (b-NH 3 ) & Pyroglutamic Acid -17 Da Q to q

21. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 21 G S/E S\G\I\F\T\N/T K Deamidation G S/E/S|G|I|F|T|D\T K 6.62 43.4% +0.986 Da G S/E/S|G|I|F|T|n\T K 18.35 96.9% +0.007 Da

22. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 22 Deamidation of Asn +1Da ionsource.com Asn –NH + O = Asp

23. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 23 CNHO +43Da Carbamylation from Urea in Digest Buffer +43Da

24. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 24 +43 b ions Carbamylated N-term I/G/E|G/T/y/G V|V|Y\K P(m/z)-CNHO P(m/z)-CNHO-H 2 O

25. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 25 8.78 71.0%  FwdRev: 3.49 (K)E E m E S A E G|L|K\G P/m\K(S) Top Database Search Result Merged 4 spectra same precursor 50 sec window different peptides Know Your Chromatographic Peak Widths

26. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 26 Related Proteins : Distinct Non-differentiable Peptides (R)N P P R\F A\F|V|E|F|E|D|P\R(D) (R)R G G/P P\F A\F|V|E|F|E|D|P R(D)

27. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 27 Frequency of Dominance at Adjacent AA’s – v9, z=2 MobilePartially Mobile Non-mobile # dominant ions # total cleavages >0.8 0.4 - 0.8 0.1 - 0.4 - (<3 obsv) 4525 spectra 2061 spectra 114 spectra

28. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 28 Precursor z=2, 6699 spectra from a trypsin GeLC/MS/MS experiment on an LTQ-FT Proton Mobility Mobile:z pre > #Arg + #Lys + #His Partially mobile:z pre #Arg Non-mobile:z pre < #Arg Frequency and Distribution Dominant Ions v9 67% 72% 76% 5758 2974 177

29. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 29 Position-dependent Dominant Ions (K)V/A|E|I/E|H|A\E\K(E) y ++ -h 2 o @ y n-2 E at position 3

30. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 30 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 Short Peptides Often Yield a Dominant Ion Cleavage Between Residues 2 & 3 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. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 31 Dominant Ions – Mobile b2/yn-2 v25 (K)A N|S/N/L/V L|Q|A|D\R(S) b 2 /y n-2

32. Karl Clauser Proteomics and Biomarker Discovery 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. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 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  =0.03638 F~m (C9 H9 N O, C5 H9 N O S) 147.06841 ~ 147.0354  =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  =0.03638 DA~W~VS (C7 H10 N2 O4, C11 H11 N2 O, C8 H14 N2 O3) 186.06405 ~ 186.07931 ~ 186.10044  =0.01526  =0.02113

34. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 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. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 35 Acknowledgements Broad Institute Steve Carr Terri Addona Jinyan Du Phillip Mertins MIT Michael Yaffe Majbrit Hjerrld Drew Lowery

36. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 36 Near Self Reversal21.16 0.0098.6 M205m FNADEFEDmVAEK FKAVmDEFEDAnK

37. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 37

38. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 38

39. Karl Clauser Proteomics and Biomarker Discovery 10/14/2015 9:47:49 AM 39 (K) L/G|F/s/L T/P/S K (G) (K) L/G|F/S/L t/P/S K (G) 10.37 10.60