1. Creation of assays using repositories

2. Overview Spectral libraries Overview of public repositories for SRM
Selecting peptides
Creating assays
Sharing assays

3. Spectral libraries MS/MS spectra, identified with high confidence
Which peptides are detectable by MS, and which fragments are seen?
Primary usage: Spectral searching
Main resources: NIST, PeptideAtlas (the later also hosts NIST libraries) and GPMDB
Typically not QQQ spectra!

4. PeptideAtlas Peptide identifications from MS/MS.
All data processed through the Trans Proteomic Pipeline (TPP)
PeptideProphet to derive a probability for the correct identification for all peptides
Protein view with identified peptides.
Focus on finding proteotypic peptides
Also used to create spectral libraries
Extending with SRM/MRM atlas. Annotated transitions for peptides.

5. Peptide atlas protein view

6. Best peptides?

8. Selecting peptides for SRM using Peptide Atlas
Observed peptides and predictions
Tested transitions

9. Spectrum view

10. Some published transition lists

11. To come: www.srmatlas.org
Press release at ETH home page:

12. GPMDB Stores peptides identified by searches in GPM (X!Tandem)
Protein coverage from different searches are stored.
SNPs and post-translational modifications
Contents used by the X!Hunter spectral searching algorithm
Also provides proteotypic peptides
Provides fixed annotation with GO, BTO

13. Coverage from the GPM DB

14. Using The GPMDB for designing transitions
Search for favourite protein – Accession number is not Uniprot! Keyword or gene identifier.

15. Select peptides

20. And another one: www.mrmaid.info

21. How good are peptide predictions?
96 human transcription factors
Whole protein synthesized and digested
Of the optimal proteotypic peptides only 18% present in NIST spectral library
Predictions of proteotypic peptides just slightly better than random

22. Sharing transition lists
Transition lists are in different formats for different instruments.
No metadata!

23. TraML (www.psidev.info/traml)
For exchange of transition lists
Instrument independent.
Info about settings, software, algorithms etc.

25. Todays exercise 1) Assay design using online resources
Download spectral library to use in Skyline
Select a protein of choice in the TCA cycle
-> Start at KEGG
Use Peptide Atlas and GPMDB to select peptides and transitions for the selected protein
Also perform for one of the proteins that you’ve already got transitions for.
2) Refine transition list