De Novo Sequencing v.s. Database Search

Bin MaSchool of Computer Science

University of WaterlooOntario, Canada

de novosequencing

1

2

Two Computational Approaches

protein DBdatabase search

MS/MS Spectra

peptidespeptides

Is This a True Peptide Identification?

Take Advantage of Both Approaches

DB search

Assigned?

Yes

NoDe Novo

All Spectra

DB peptidesnovel peptides

Facts and Fallacies

• True. It can be regarded as a “universal” DB search.

1. De novo is harder.

2. De novo is slow.• False. PEAKS processes 15 spectra/sec on a PC.

3. De novo is less reliable.• Depends.

Goals of This Talk

• A “fair” comparison• A “real” combination

What Is a Fair Comparison?

• The ability to report a long sequence tag – long enough to uniquely identify the correct peptide from

the same database.

Experiment 1.

• Purpose: to check the length of correct de novo tags on good quality spectra.

• Trypsin digest of Human tumour cell; LTQ-Orbitrap CID.

• Mascot DB search; 1% FDR.• Compare PEAKS de novo sequences with DB

search results.

Distribution of Correct Tag Length

≥5 ≥6 ≥7 ≥8 ≥9 ≥100%

10%20%30%40%50%60%70%80%90%

100%

# of correct amino acid found by PEAKS de novo sequencing

perc

atag

e in

Mas

cot h

igh

confi

dede

nt P

SM

“De Novo Only” Peptides

Experiment 1 Conclusion.

• De novo sequencing performs well in terms of finding long sequence tags.

• De novo also finds “de novo only” peptides.

Experiment 2.• Purpose: To test de novo tags’ ability for identifying the

peptides from the database?• For each de novo sequence, reports the “best-matching”

peptide from the database.• Use target-decoy to determine the FDR.

Experiment 2 Conclusion

• De novo tags have better and complementary ability in selecting the correct peptides from DB.

• This might be due to the choice of the software.

Experiment 3. De Novo Assisted DB Search# matched amino acidsbetween de novo & DB search

x+4ybest separation line

Mascot Score

0 500 1000 1500 2000 2500 3000 3500 40000.0%

0.5%

1.0%

1.5%

2.0%

2.5%

# of PSM

FDR

Mascot PEAKS DB

PEAKS DB

Conclusion

De novo both helps to improve DB search, and reports novel peptides.

DB search

Found?

De Novo

Improved DB search results

De novo only peptides

improves

no

yes

MS/MS Spectra

This is the default workflow of PEAKS 5.3.