PepArML: A model-free, result-

combining peptide

identification arbiter via

machine learning

PepArML: A model-free, result-

combining peptide

identification arbiter via

machine learning

Xue Wu, Chau-Wen Tseng, Nathan Edwards

University of Maryland, College Park, andGeorgetown University Medical Center

2

Comparison of Search Engines

• No single score is comprehensive

• Search engines disagree

• Many spectra lack confident peptide assignment

• Many spectra lack any peptide assignment

Searle et al. JPR 7(1), 2008

38%

14%28%

14%

3%

2%

1%

X! Tandem

SEQUESTMascot

3

Black-box Techniques

• Significance re-estimation• Target-Decoy search• Bimodal distribution fit

• Supervised machine learning• Train predictors on synthetic datasets• Select and/or create (many) good features

• Result combiners• Incorrect peptide IDs unlikely to match• Significance re-estimation• Independence and/or supervised model

4

PepArML

• Unified machine learning result combiner• Significance re-estimation too!

• Model-free feature use and result combination• Use agreement and features if useful

• Unsupervised training procedure• No loss of classification performance

5

PepArML Overview

X!Tandem

Mascot

OMSSA

Other

PepArML

6

PepArML Overview

X!Tandem

Mascot

OMSSA

Other

PepArML

Feature extraction

7

Dataset Construction

T),( 11 PS

F),( 21 PS

T),( 12 PS

X!Tandem Mascot OMSSA

T),( mn PS

……

8

Dataset Construction

• Calibrant 8 Protein Mix (C8) • 4594 MS/MS spectra (LTQ)• 618 (11.2%) true positives

• Sashimi 17mix_test2 (S17)• 1389 MS/MS spectra (Q-TOF)• 354 (25.4%) true positives

• AURUM 1.0 (364 Proteins)• 7508 MS/MS spectra (MALDI-TOF-TOF)• 3775 (50.3%) true positives

9

PepArML Machine Learning

• Machine learning (generally) helps single search engines

• PepArML result-combiner (C-TMO) improves on single search engines

• Sometimes combining two search engines works as well, or better, than three

10

PepArML vs Search Engines (C8)

11

True vs. Est. FDR (C-TMO, C8)

12

PepArML vs Search Engines (C8)

13

PepArML Pairs vs PepArML (C8)

14

Sensitivity Comparison

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

(10%, C8) (10%, S17) (10%, AURUM)

Classifier (FDR, dataset)

Sen

siti

vity

C-TMO

C-TM

C-TO

C-MO

C-T

C-M

C-O

Tandem

Mascot

OMSSA

15

Feature Evaluation

0

0.2

0.4

0.6

0.8

Info

Gai

n

C8

0

0.2

0.4

0.6

0.8

Info

Gai

n

S17

0

0.2

0.4

0.6

0.8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Info

Gai

n

AURUM

1 Peptide length2 hyperscore3 precursor mass delta4 # of matched y-ions5 # of matched b-ions6 # of missed cleavages7 sum matched intensity8 E-value9 sentinel

10 score11 precursor mass delta12 # of matched ions13 # of matched peaks14 # of missed cleavages15 E-value16 sentinel17 p-value18 # of matched ions19 E-value20 sentinel

Ta

nde

mO

MS

SA

Ma

sco

t

16

Application to Real Data

• How well do these models generalize?

• Different instruments• Spectral characteristics change scores

• Search parameters• Different parameters change score values

• Supervised learning requires• (Synthetic) experimental data from every instrument• Search results from available search engines• Training/models for all

parameters x search engine sets x instruments

17

Model Generalization

Train C8 / Score S17

Train S17 / Score S17

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Rescuing Machine Learning

• Train a new machine learning model for every dataset!• Generalization not required• No predetermined search engines, parameters,

instruments, features

• Perhaps we can “guess” the true proteins• Most proteins not in doubt• Machine learning can tolerate imperfect labels

19

Unsupervised Learning

20

Unsupervised Learning (S17)

21

Unsupervised Learning (S17)

22

Protein Selection Heuristic

• Modeled on typical protein identification criteria• High confidence peptide IDs• At least 2 non-overlapping peptides• At least 10% sequence coverage

• Robust, fast convergence

• Easily enforce additional constraints

23

What about real data?

Dr. Rado Goldman (LCCC, GUMC)• Proteolytic serum peptides from clinical

hepatocellular carcinoma samples• ~ 200 MALDI MS/MS Spectra (TOF-TOF)

PepArML for non-specific search of IPI-Human

• Increase in confidence & sensitivity• Observation of “ragged” proteolytic trimming

24

Protein Identification Example

M T OKey E-value

< 1e-5< 0.05Any IDNo ID

*

25

Future Directions

• Apply to more experimental datasets• Integrate

• novel features• new search engines, spectral matching• multiple searches with varied parameters,

sequence databases• Construct meta-search engine• FDR by bimodal fit instead of decoys• Release as open source

• http://peparml.sourceforge.org

26

http://PepArML.SourceForge.Net

27

Acknowledgements

• Xue Wu* & Dr. Chau-Wen Tseng, • Computer Science

University of Maryland, College Park• Dr. Brian Balgley, Dr. Paul Rudnick

• Calibrant Biosystems & NIST• Dr. Rado Goldman, Dr. Yanming An

• Department of OncologyGeorgetown University Medical Center

• Kam Ho To• Biochemistry Masters student

Georgetown University

• Funding: NIH/NCI CPTAC

28

29

PepArML vs Search Engines (S17)

30

PepArML vs Search Engines (S17)

31

PepArML Pairs vs PepArML (C8)

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PepArML Pairs vs PepArML (S17)

33

PepArML Pairs vs PepArML (S17)

34

Unsupervised Learning (C8)

35

Unsupervised Learning (C8)