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, 2009
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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
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3. AA structures K H R 128 137 156 D E 115 129 N Q 114 128 S T Y
pK: N-term 7.5
pK: C-term 3.5
K  H R
pK:
D E
pK:
N Q
S T Y P 97
M C
(+57 IAA)
L I
G A V
F W
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4. Charge-directed Fragmentation Scheme
zHz+ O O O O +
H2N CH C NH CH C NH CH C NH CH C OH R1 R2 R3 R4 H
b ion formation
and/or
y ion formation
H2N CH C NH CH C NH CH C R1 R2 O R3
NH CH C OH R4 O H + b3 + y1 +
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. H2N CH C NH CH C NH CH C NH CH C OH
Sequence Specfic Fragment Ion Types a3 b3 c3
nHn+
H2N CH C NH CH C NH CH C NH CH C OH R1 R2 R3 R4 O 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 1

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
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7. Dual Picket Fence A E/D|T|A|L|Y|Y|C A\K 163 163 113 71 101 115
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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,
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9. Diagnose Doubly Charged Fragment Ions
I/A|D|A|H|L|D|R
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10. Dominant Cleavage Proline N-side
N F|P/S/P V D A A F R y9 b2 28
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11. Sparse Dominant Fragmentation
(K)I S R|P G D|S D|D|S R(S)
Non-mobile proton
zpre < #Arg
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12. Cry Babies (b-H2O & b pairs)
P(m/z)-H2O
P(m/z)-2H2O
E/H/A|V/E|G/D|C D|F Q L L K
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13. Interpreting MS/MS Spectra is Fun!!
Kaitlin
Aidan
Jack
Andrea
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14. 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.
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15. Phospho Site Ambiguity – S/T
L P/S s/P/V|Y/E/D|A A S F K
P(m/z)-H3PO4-H2O
P(m/z)-H3PO4
P(m/z)-H3PO4-2H2O
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16. 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
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17. 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 yes yes no
2004 MCP, 3, digest, SCX
LC/MS/MS
Rush, J, … Comb, MJ digest lysate LCQ Deca XP yes no no
2005, Nat Biotech, 23, pTyr Ab
LC/MS/MS
Collins, MO, …Grant, SGN protein IMAC Q-Tof Ultima no yes no
2005, J Biol Chem, 280, peptide IMAC
LC/MS/MS
Gruhler, A, … Jensen, ON digest lysate LTQ-FT yes no no
2005 MCP, 4, SCX, IMAC
LC/MS/MS
“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.”
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18. Expect Woes & Nuisances
Sample Handling Chemistry
Carbamylation +43 nterm, Lys urea in digest buffer
Deamidation 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
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19. Stinkers (b-NH3) & Pyroglutamic Acid
-17 Da
Q to q
(R)Q L/Q/L/A|Q/E/A|A Q\K(R)
P(m/z)-NH3
(R)q L/Q|L|A|Q|E|A|A\Q\K(R)
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20. Deamidation G S/E/S|G|I|F|T|n\T K G S/E/S|G|I|F|T|D\T K
18.35
96.9%
+0.007 Da
G S/E/S|G|I|F|T|D\T K
G S/E S\G\I\F\T\N/T K
6.62
43.4%
+0.986 Da
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21. Deamidation of Asn +1Da Asn –NH + O = Asp ionsource.com

22. Carbamylation N/S/L/E/T/L/L|y/K|P V/D\R V/S T A/Q/D V/I|Q Q t L\C K
+43
18.4
89%
V/S T A/Q/D V/I|Q Q t L\C K +0
11.1
68% +0
18.5
93%
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23. Carbamylation from Urea in Digest Buffer +43Da
CNHO +43Da
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24. Carbamylated N-term I/G/E|G/T/y/G V|V|Y\K +43 b ions P(m/z)-CNHO
P(m/z)-CNHO-H2O
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25. Met Oxidation – localizing the site
(R)G V D L D Q L/L|D|M|S|Y|E|Q|L|m|Q|L/Y S A R(Q)
(R)G V D L D Q L/L|D|m|S/Y/E|Q|L|M|Q|L/Y S A R(Q)
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26. 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
Merged 4 spectra
same precursor
50 sec window
different peptides
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27. 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) =
K ~ Q (C6 H12 N2 O, C5 H8 N2 O2) ~ D =
F~m (C9 H9 N O, C5 H9 N O S) ~ D =0.0330
Isobaric AA combinations
GG=N (C4 H6 N2 O2 , C4 H6 N2 O2)
GA=Q~K (C5 H8 N2 O2, C5 H8 N2 O2, C6 H12 N2 O) ~ D =
DA~W~VS (C7 H10 N2 O4, C11 H11 N2 O, C8 H14 N2 O3) ~ ~ D = D =
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28. Additional Resources Google: “de novo sequencing tutorial”
Don Hunt and Jeff Shabanowitz - manual
Rich Johnson - manual
PEAKS - automated
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29. 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

30. Frequency of Dominance at Adjacent AA’s – v9, z=2
Mobile
Partially Mobile
4525 spectra
2061 spectra
# dominant ions
# total cleavages
>0.8
- (<3 obsv)
Non-mobile
114 spectra
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31. Frequency and Distribution Dominant Ions v9
67%
72%
76%
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
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32. 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.
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33. Acknowledgements Broad Institute Steve Carr Terri Addona Jinyan Du MIT
Michael Yaffe
Majbrit Hjerrld
Drew Lowery
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34. Frequency of Position Dependent Dominant Ion(s) v9
Proton Mobility
Mobile: zpre > #Arg + #Lys + #His
Partially mobile: zpre < #Arg + #Lys + #His and > #Arg
Non-mobile: zpre < #Arg
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35. 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)
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36. Setting Autovalidation Thresholds
Step 1 - Protein Mode
Step 2 - Peptide Mode
2 or more peptides/protein
Each spectrum: moderate or better score
1 peptide/protein
Each spectrum: excellent score
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37. Distinguishing 7 Family Members
( proteins)
13.4 YWHAZ
16.5 YWHAE
13.0 SFN
11.1 YWHAB
11.4 YWHAT
13.3 YWHAG
13.5 YWHAH
Gene
Symbol
Wt/Mut
PBD
None have a PLK-PBD binding motif: S[st]P
Each has 2-4 PLK phosphorylation motifs: [ED]X[st][FLIYWVM]
No phosphorylated peptides were recovered.
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38. Chromatographic Peak Sampling: Abundance/Identity Trade-offMS
Relative Abundance
Periodic Focus on
Several Peptides
Abundance
MS/MS
Sliding Focus on
Each Peptide
Identity
Retention Time
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39. Distinguishing Family Members
(ROCK1 & ROCK2 )
#Same #Distinguishing WT/Mut Gene
Peps Peps PBD Symbol
ROCK2
ROCK1
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40. Enabling Integrated Reverse Database Searches
Each database sequence candidate passing the parent mass filter is additionally subject to “inner sequence reversal” and interpreted against the MS/MS spectrum.
i.e.
SAMPLER
Becomes
SELPMAR
Search time increases ~1.5X
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41. Reviewing Reversed Database Search Details
This is an overview of the SpectrumMill software. The next several slides show some of these features in more detail.
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42. Near-Identical Sequences Found in Reversed DB
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43. Experimental Spectrum theoretical fragment ions Experimental Spectrum
SEQUEST - preliminary search Pm
Sequence
Database
Experimental Spectrum
Relative Abundance
Step 1
calculate ALL
theoretical fragment ions
for EACH sequence
Step 3
Mass (m/z)
Step 2
Filter
Compare Pm Pm b y b y b y b y
Filtered
Experimental Spectrum
Relative Abundance
Model Spectrum a a a a
Relative Abundance
b-NH3
y-NH3
b-NH3
y-NH3
b-NH3
y-NH3
b-NH3
y-NH3
b-H2O
y-H2O
b-H2O
y-H2O
b-H2O
y-H2O
b-H2O
y-H2O
Mass (m/z)
Mass (m/z)

44. MS-Tag preliminary search
Sequence
Database Pm
Filtered
Experimental Spectrum
Relative Abundance
Step 2
calculate partial ladders
for EACH sequence
Mass (m/z)
Step 3
Step 1
Transform b
b-NH3
b-H2O a
Compare b
b-NH3
b-H2O a Pm Pm b
b-NH3
b-H2O a
N-terminal
Sequence Spectrum
Relative Abundance
Partial N-terminal Ladder
Relative Abundance
Mass (m/z) Pm
Mass (m/z) Pm
y-NH3 y
y-NH3 y
y-NH3
C-terminal
Sequence Spectrum
Relative Abundance
Relative Abundance
Partial C-terminal Ladder y
Mass (m/z)
Mass (m/z)

45. Mass Differences Correspond to Amino Acidsu q e s e q u e
Intensity n n c e e e q c s n e u s e c e
m/z
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46. Graphy Theory Based de novo Algorithms
vertices (from peak m/z’s)
edges (from mass differences)
Transform to
Spectrum Graph
Find best path
Sequence
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47. SpectrumMill Scoring of MS/MS Interpretations Non-assignment Penalty
(R)E F E|I|I|W|V T K(H)
9.24
78.1%
DEBS: -3
DFwdRev: 1.75
Score =
Assignment Bonus
(Ion Type Weighted) -
Non-assignment Penalty
(Intensity Weighted)
Peak Selection: De-Isotoping, S/N thresholding,
Parent - neutral removal, Charge assignment
Match to Database Candidate Sequences
SPI (%)
Scored Peak Intensity
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48. Peptide Sequencing y3 y2 y1 y2 - NH3 y3 -H2O b2-H2O b3- NH3 a2 b2 a3
HO NH3+
| |
R O R2 O R O R4
| || | || | || |
H -- N --- C --- C --- N --- C --- C --- N --- C --- C --- N --- C -- COOH
| | | | | | |
H H H H H H H y3 y2 y1
y2 - NH3
y3 -H2O
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49. Cry Babies (b-H2O w/o b) No b/y pairs E/C|L/Q/T/C/R 113 128 101 160
P(m/z)-H2O
P(m/z)-2H2O No
b/y
pairs
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