1. Facts and Fallacies about de Novo Sequencing & Database Search

2. 1. There are a large number of high quality spectra left unassigned after DB search. True False

3. Unassigned Spectra in ABRF/iPRG 2011 Study

4. Unassigned Spectra Nonspecific trypsin cleavages Novel peptide/incomplete database PTM Mutations PEAKS PTM SPIDER PEAKS DB De novo sequencing

5. 2. Nonspecific cleavage, PTM, mutations and novel peptides are the main reasons for the unassigned spectra. True False

6. Average Software Misses Peptides

7. 3. De novo sequencing is slow. True False

8. Speed PEAKS 6 de novo sequence 15 spec/second. – Intel i7 Quad Core, 8GB RAM. – Trypsin – Orbitrap CID MS/MS, mostly charge +2/+3 PEAKS 7 (coming soon): – Improve speed on high charge states and longer peptides. – Add 8 core support in standard (desktop) license.

9. 4. De novo should be done after DB search. True False DB search DB peptides de novo seq. Unassigned spectra de novo peptides

10. Order of de Novo and DB Better conduct de novo on all spectra. – De novo not slow, and computing is cheap. – De novo provides independent validation for DB result. # consensus AA (de novo vs. DB search) true score false without de novo with de novo

11. 5. My protein sequence is confirmed with two unique peptide hits. True False

13. Routine Full Protein Coverage For regular proteins, full sequence coverage can be routinely achieved with – 3 or more enzyme digests, and – multiple algorithms in PEAKS 6. For highly variable proteins (such as antibodies), BSI offers data analysis service for antibody sequencing.

14. 6. If a peptide is identified with 1% FDR, then it’s sequence is 99% correct. True False

15. Peptide Validation vs. Amino Acid Validation You are confident about the peptide sequence only if you can de novo sequence it, and the de novo sequence matches the database peptide.

16. 7. I don’t need de novo sequencing if I have a protein DB. True False

17. 8. Target-decoy provides a reliable result validation for every DB search engine. True False

18. weak hits confident protein weak protein Target-Decoy Incompatible with Certain Highly Optimized Search Engines Adding “protein bonus” to peptide hits increases accuracy. But it creates bias between target and decoy. – In extreme, bonus is so large that only peptides from target proteins are selected. – This gives the wrong impression that FDR=0, while there are still false peptides in the result.

19. weak hits confident protein weak protein Decoy Fusion Is A More Powerful Validation Method Decoy fusion append a decoy sequence to each protein. Recreates the balance. The built-in validation method since PEAKS 5.3.

20. 9. Combining 1% FDR results of multiple engines gives 1% FDR. True False

21. Error Accumulation In PEAKS, the inChorus algorithm automatically selects a less than 1% common FDR for each engine so that the combined FDR is approximately 1%. PEAKS DBMascot 1696(37) 2.4% 2174(1) 0.1% 195(22) 13% Target(decoy) FDR% PEAKS DB 3870(38) 1% 2369(23) 1% Mascot Correct < sum of the two Error ≈ sum of the two Correct < sum of the two Error ≈ sum of the two Combined FDR = 1.5%

22. 10. There is no automated way to validate de novo sequencing results. True False