1. De Novo Sequencing v.s. Database Search Bin Ma School of Computer Science University of Waterloo Ontario, Canada

2. de novo sequencing 1 2 Two Computational Approaches protein DB database search MS/MS Spectra peptides

4. Is This a True Peptide Identification?

5. Take Advantage of Both Approaches DB search Assigned? Yes No De Novo All Spectra DB peptides novel peptides

6. 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.

7. Goals of This Talk A “fair” comparison A “real” combination

8. 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.

9. 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.

10. Distribution of Correct Tag Length

11. “De Novo Only” Peptides

12. Experiment 1 Conclusion. De novo sequencing performs well in terms of finding long sequence tags. De novo also finds “de novo only” peptides.

13. 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.

14. 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.

15. Experiment 3. De Novo Assisted DB Search # matched amino acids between de novo & DB search x+4y best separation line Mascot Score

16. PEAKS DB

17. 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.