Karl ClauserProteomics and Biomarker Discovery 1

Taming Errors for Peptides with Post-Translational Modifications

Bioinformatics for MS Interest GroupASMS June 16, 2014

Baltimore, MD

Karl ClauserBroad Institute of MIT and Harvard

Karl ClauserProteomics and Biomarker Discovery 2

Making Inaccurate FDR Estimates

301

12

k Sites Distinct Peptides (CI#)

k Sites Distinct Peptides False (#)

14,995

64

All Distinct Peptides (CI#)

All Distinct Peptides False (#)

FDR: 0.85% FDR: 7.97%

2% Lys-AcUnenriched

32499

234 201

s|t|y Sites Distinct Peptides (CI#)

s|t|y Sites # Spectra False

35181

228

All Dis-tinct Pep-tides (CI#)

All Dis-tinct Pep-tides False (#)

FDR: 1.30% FDR: 1.24%

92% PO4 styIMAC -enriched

Karl ClauserProteomics and Biomarker Discovery 3

Variable Modifications Expand the Search Space

Candidates passing precursor mass filterPrecursorMH+ shift

AAcomposition

-256 -176 -160 -97 -81 -80 -32 -16 -2 -1 0 17

3ST 2ST 2ST 1ST 1ST 1ST 2M 1M 2N 1N * ^Q1M 1M 1M 1N? ? ? ? ? ? ? ? ?

FixedModsonly

AllowVariable

Mods

CalculateMH+ fixed mods only

Calculate MH+Variable modcombinations

tolerancefilter

Shift rangefilter

AA compositionfilter

tolerancefilter

precursor mass filter

Total candidates = (S M0) + (S M1)N + (S M2)2N + (S M16)M + (S M32)2M + (S M80)ST + …

Relative # candidates: (S M0) > (S M80)ST > (S M1)N >> (S M1)M > (S M1)^Q

(S M80)STY > (S M160)2STY > (S M240)3STY

Karl ClauserProteomics and Biomarker Discovery 4

FDR across Multiple Experiments or Replicates

85805

2651

s|t|y Sites Distinct Peptides (CI#)

s|t|y Sites Distinct Peptides False (#)

FDR: 1.30%

92% PO4 styIMAC -enriched

32499

234 201

s|t|y Sites Distinct Peptides (CI#)

s|t|y Sites # Spectra False

FDR: 1.30% FDR: 6.18%

True peptides tend to repeatFalse peptides tend to not

True spectra/peptide: 2.3 28.1False spectra/peptide: 1.2 3.0

1 experiment 36 experiments

Karl ClauserProteomics and Biomarker Discovery 5

Where can we make Improvements?To observe precursor must enrich

concentrateionize

To select precursor must observe precursorhave time

To fragment must select precursor

To identify must fragment

To localize must cleave backbone

Enrichment peptide/proteinDigestion enzymeChemical label

Chromatographic resolutiongradient timeparticle size (<2 micron)

Choose Precursor m/zm/z, delta m/zabundancechargetime – chromatographic apex

Choose Dissociation mode: CID,HCD, ETD

Combine precursor ions before MS/MS+2, +3, +4Light, medium, heavySWATH, DIA

Combine fragment ions before mass analyzing+2, +3, +4CID,HCD, ETD

Combine spectra to interpret, localize+2, +3, +4CID, HCD, ETD

ReferencePatient Specific Sequence Databases (RNA-Seq, Whole Exome)Spectral libraries (dissociation mode, chemical label, organism)

Error rateFDR: match subsets for score threshold, reportingFLR: widely used metric

Sample PreparationData Acquisition HardwareData Acquisition Software ControlData Interpretation

Karl ClauserProteomics and Biomarker Discovery 6

y-H3PO4 or y-H2O Ions?

H2O

H3PO4 (R)S\T\P L\T L E I s/P D/N S L/R(R) (R)S\T\P L\t L E I S/P D/N S L/R(R)

Karl ClauserProteomics and Biomarker Discovery 7

y-H3PO4 not y-H2O IonsH3PO4 (R)S\T\P L\T L E I s/P D/N S L/R(R)

+2precursor

+3precursor

(R)S T\P L\T\L\E I s/P/D/N S L/R(R)

b6

757.5

+2

+3

Karl ClauserProteomics and Biomarker Discovery 8

Performance of Spectrum Mill ID/Localization Algorithm Revisions

SM_35_NLPpref (neutral loss of phosphate preferred)Distort fragment ion type scoresSo that2 H3PO4 loss beats2 H2O loss

For alternate localizationsshould H3PO4 loss ions bepreferred to H2O loss?

Recover accompanyingb/y ions by decreasing CE?

Is this more an iTRAQ issue or an HCD feature?

Comparing different parent charge MS/MS of samepeptide very helpful.

SM_35Score: y- H3PO4 = y-H2OScore: b- H3PO4 = b-H2O

NPA: no possible ambiguity (only 1 STY in peptide)