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De Novo Sequencing v.s . Database Search

De Novo Sequencing v.s . Database Search. Bin Ma School of Computer Science University of Waterloo Ontario, Canada. protein DB. Two Computational Approaches. MS/MS Spectra. 1. 2. database search. de novo sequencing. peptides. peptides. Is This a True Peptide Identification?.

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De Novo Sequencing v.s . Database Search

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

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

  3. Is This a True Peptide Identification?

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

  5. Facts and Fallacies 1. De novo is harder. • True. It can be regarded as a “universal” DB search. 2. De novo is slow. • False. PEAKS processes 15 spectra/sec on a PC. 3. De novo is less reliable. • Depends.

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

  7. 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.

  8. Experiment 1. • Purpose: to check the length of correct de novo tags on good quality spectra. • Trypsin digest of Human tumour cell; LTQ-OrbitrapCID. • Mascot DB search; 1% FDR. • Compare PEAKS de novo sequences with DB search results.

  9. Distribution of Correct Tag Length

  10. “De Novo Only” Peptides

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

  12. 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.

  13. 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.

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

  15. PEAKS DB

  16. Conclusion MS/MS Spectra De novo both helps to improve DB search, and reports novel peptides. improves This is the default workflow of PEAKS 5.3. De Novo DB search no Found? yes De novo only peptides Improved DB search results

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