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Creation of assays using repositories

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Presentation on theme: "Creation of assays using repositories"— Presentation transcript:

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?

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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

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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.

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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


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