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 ClauserProteomics and Biomarker Discovery

04/19/23 10:38 PM 1

Manual De Novo Peptide MS/MS InterpretationFor Evaluating Database Search Results

Karl R. ClauserBroad Institute of MIT and Harvard

Cold Spring Harbor Proteomics CourseJuly, 2010


04/19/23 10:38 PM 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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AA Structures & Masses

http://ionsource.com/Card/clipart/aaclipart.htm

G A V57 71 99

S T Y87 101 163

F W 147 186

P97

M C131 103 (+57 IAA)

L I113 113

D E115 129

pK: 4.0 4.5pK: C-term 3.5

K H R128 137 156

N Q114 128

pK: 10 6 12 pK: N-term 7.5

Name AA MassGly G 57Ala A 71Ser S 87Val V 99Thr T 101Leu/Ile L/I 113Asn N 114Asp D 115Lys/Gln K/Q 128Glu E 129Met M 131His H 137Phe/Met-ox F/m 147Arg R 156Cys-IAA C 160Tyr Y 163Trp W 186


H2N CH C NH CH C NH CH C NH CH C OH

R1 R2 R3 R4

O O O O

H

b3

b ion formation

NH CH C OH

R4

O

H

+H y1

y ion formation

Charge-directed Fragmentation Scheme

+Neutral pumped away by vacuum system

and/or

H2N CH C NH CH C NH CH C

R1 R2

O O O

R3

zHz+

+

+Neutral pumped away by vacuum system

+

Proton Mobility

Mobile: zpre > #Arg + #Lys + #HisPartially mobile: zpre < #Arg + #Lys + #His and > #ArgNon-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, EN-term side of P


H2N CH C NH CH C NH CH C NH CH C OH

R1 R2 R3 R4

O O O O

y1

b3

nHn+

Sequence Specfic Fragment Ion Types

z1x1

a3 c3

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


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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


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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


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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

101000

10 510 710 9

10 1110 1310 1510 1710 1910 2110 2310 2510 2710 2910 3110 33

Num

ber

of P

eptid

es

1 5 9 13 17 21 25

All Sequences (Permutations 20 N)

Peptide Length (N)

All AA Compositions (Combinations)Sequences in Human Genome

multiple copiesof eachsequence present 1 sequence / composition

may be present

log scale

(# of genes) x (mean length - N)(100,000) x (350aa - N)

N < 6N > 11


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I/A|D|A|H|L|D|R

Diagnose Doubly Charged Fragment Ions


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Dominant Cleavage Proline N-side

N F|P/S/P V D A A F R y9

b2

28 87 97


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Sparse Dominant Fragmentation

115 202

202 115

(K)I S R|P G D|S D|D|S R(S)

Non-mobile protonzpre < #Arg


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E/H/A|V/E|G/D|C D|F Q L L K

Cry Babies (b-H2O & b pairs)

P(m/z)-H2O

P(m/z)-2H2O


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Interpreting MS/MS Spectra is Fun!!

Kaitlin

Aidan Jack

Andrea


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Interpreting MS/MS Spectra is Fun!!

Kaitlin

Aidan Jack

Andrea


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MajorDatabase

Peptide not in database. Mutation. MS/MS not from a peptide.

Unanticipated Protein ChemistryChemical modification, post-translational modification.

Enzyme/Ion SourceNon-specific cleavage. In-source fragmentation yields MS3.

MinorAlgorithm

Fragment ion types of instrument not accounted for. Peak Detection.

Instrument ResolutionWrong parent charge. Wrong fragment charge.

User CompetenceWrong parameters selected.

Source of Incorrect MS/MS Interpretations


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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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Phospho Site Ambiguity – S/TL 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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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 no2004 MCP, 3, digest, SCX1093-1101 LC/MS/MS

Rush, J, … Comb, MJ digest lysate LCQ Deca XP 628 0 yes no no2005, Nat Biotech, 23, pTyr Ab94-101 LC/MS/MS

Collins, MO, …Grant, SGN protein IMAC Q-Tof Ultima 331 42 no yes no2005, J Biol Chem, 280, peptide IMAC5972-5982 LC/MS/MS

Gruhler, A, … Jensen, ON digest lysate LTQ-FT 729 0 yes no no2005 MCP, 4, SCX, IMAC310-327 LC/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.”


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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

Expect Woes & Nuisances


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(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)-NH3

Stinkers (b-NH3) & Pyroglutamic Acid

-17 Da

Q to q


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G S/E S\G\I\F\T\N/T K

Deamidation

G S/E/S|G|I|F|T|D\T K

6.6243.4%

+0.986 Da

G S/E/S|G|I|F|T|n\T K

18.3596.9%

+0.007Da


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Deamidation of Asn +1Da

ionsource.com

Asn –NH + O = Asp


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CNHO +43Da

Carbamylation from Urea in Digest Buffer +43Da


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+43b ions

Carbamylated N-term

I/G/E|G/T/y/G V|V|Y\K

P(m/z)-CNHO

P(m/z)-CNHO-H2O


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8.78 71.0%

FwdRev: 3.49

(K)E E m E S A E G|L|K\G P/m\K(S)

TopDatabaseSearchResult

Merged 4 spectrasame precursor50 sec window

different peptides

Know Your Chromatographic Peak Widths


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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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Frequency of Dominance at Adjacent AA’s – v9, z=2

Mobile Partially 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


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Precursor z=2, 6699 spectrafrom a trypsin GeLC/MS/MS experiment

on an LTQ-FT

Proton Mobility

Mobile: zpre > #Arg + #Lys + #HisPartially mobile: zpre < #Arg + #Lys + #His and > #ArgNon-mobile: zpre < #Arg

Frequency and Distribution Dominant Ions v9

67%

72%

76%

5758

2974

177


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Position-dependent Dominant Ions

(K)V/A|E|I/E|H|A\E\K(E) y++-h2o @ yn-2 E at position 3

% Frequency # Dominant # Total Possible Mean Intensity RatioIon Type M PM NM M PM NM M PM NM M PM NMnterm E b-h2o * 37.7% 29.0% 163 47 432 162 6 3.7 3.6nterm Q b-nh3 15.7% 27.5% 30 14 191 51 1 2.9 2.9b2/yn-2 ** 14.3% 7.3% 1.8% 645 150 2 4503 2051 114 4.5 3.6 2.1bn-2 contains RHK ^ 1.2% 100.0% 14 2 16 1167 2 3.0 2.5y++-h2o @ yn-2 3(DnEST) ^^ 4.8% 1.9% 67 13 1385 699 37 4.0 3.1 y++-h2o @ yn-2 3(ST) 11.2% 1.2% 64 3 571 251 7 y++-h2o @ yn-2 3(E) 0.4% 3.3% 2 9 479 269 21y++-nh3 @ yn-2 3(QH) 3.2% 8.2% 10 19 316 231 9 2.6 3.0* n terminal residue is E, b-h2o ion occurs at any position** fragment ion occurs between residues 2 and 3, either b or y ion formed^^ b ion occurs between 2nd and 3rd from last residues, the ion contains an RHK residue^^ fragment ion occurs between residues 2 and 3, where residue 3 is D, deamidated Asn (Asp), E, S, or T


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Bonus C-side b2 residues at position 3: PRKHBonus N-side b2 residues at position 1 or 2: PRKHNQqVILFYWBonus 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.


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Dominant Ions – Mobile b2/yn-2 v25

(K)A N|S/N/L/V L|Q|A|D\R(S) b2/yn-2


Physiochemical Complications to Computational Interpretation

• Incomplete Fragmentation• Inconsistent intensity of fragment ion types

Instrument type dependentAmino 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


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Consequences of Inappropriate Tolerance Units(using Da tolerance when instrument errors are in ppm)

tooloose

tootight

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


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Additional Resources

Google: “de novo sequencing tutorial”

Don Hunt and Jeff Shabanowitz - manual http://www.ionsource.com/tutorial/DeNovo/DeNovoTOC.htm

Rich Johnson - manualhttp://www.abrf.org/ResearchGroups/MassSpectrometry/EPosters/ms97quiz/SequencingTutorial.html

PEAKS - automatedhttp://www.bioinformaticssolutions.com/products/peaks/support/tutorials/PEAKS_De_Novo.html


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Acknowledgements

Broad Institute

Steve CarrTerri AddonaJinyan DuPhillip Mertins

MIT

Michael YaffeMajbrit HjerrldDrew Lowery


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Near Self Reversal

21.1621.16

0.0098.698.6

M205m FNADEFEDmVAEKFKAVmDEFEDAnK


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(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

Documents

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