1. FDR Thresholding Caleb J. Emmons Slide: 1

2. What is FDR? Slide: 2 If decoy proteins are present Protein FDR = # decoy proteins identified # target proteins identified Peptide FDR = # spectra from decoy proteins # spectra from target proteins

3. The FDR Browser

4. How does FDR Thresholding work? Slide: 4 1 5 4 3 2 The “FDR Landscape” Minimum number of peptides

5. How does FDR Thresholding work? Slide: 5 The “FDR Landscape”

6. Some Fine Points Slide: 6 Confusing!

7. Protein Clustering Poster 509, Tuesday 10:30-1:00 Informatics: Quantification/Validation Caleb J. Emmons Slide: 7

8. What is a Cluster? Slide: 8

9. Total Peptide Evidence Slide: 9 PE tot (A) = sum of peptide probabilities over all peptides matching A ProteinPE tot K1C101481% K1C141061% K1C16852% K1C17503%

10. Joint Peptide Evidence Slide: 10 PE joint (A, B) = sum of peptide probabilities over all peptides matching A and B PE joint K1C10K1C14K1C16 K1C14184% K1C16184%661% K1C1784%375%175%

11. Proteins A and B are in the same cluster if they are directly similar, or if they can be connected with a sequence of proteins that are directly similar. Cluster Formation Slide: 11 A ≈ B if 1) their joint evidence is at least 95%, and 2) their joint evidence is at least half of the total evidence for A or B Directly similar Clusters

12. Cluster Formation Slide: 12 ProteinPE tot K1C101481% K1C141061% K1C16852% K1C17503% PE joint K1C10K1C14K1C16 K1C14184% K1C16184%661% K1C1784%375%175% A ≈ B ? K1C10K1C14K1C16 K1C14no K1C16noyes K1C17noyesno

13. Peptide-Protein Weights Slide: 13 A B C PE excl (C) = sum of peptide probabilities over all peptides exclusively matching C W(p, C) =

14. Spectrum Counting Slide: 14 SEQ1, +3SEQ1, +2SEQ2, +2 SEQ3, +2 SEQ7, +3 SEQ4,+2 SEQ5, +2 A B C Protein A 3231 Protein B 4242 Protein C 4231 Cluster of B&C 6554 Total Spectra Exclusive Spectra Unique Spectra Exclusive Unique Peptides Exclusive peptide/spectrum: associated only with this single cluster/protein Unique peptides: only consider amino acid sequences Unique spectra: only consider amino acid sequence, modifications, & charge state

15. Quantitative Values Slide: 15 Total and Weighted Spectrum Counts run over all spectra in the cluster Total Ion Current (TIC) and Precursor Intensity may be computed, treating the cluster as a collection of spectra. Normalized Spectral Abundance Factor (NSAF) roughly consists of a ratio of an exclusive spectrum count and protein length, so does not make direct sense on the level of cluster (as clusters do not have a ‘length’). However, the average NSAF over the member proteins gives an interpretable value. Similarly, we compute the Exponentially Modified Protein Abundance Index (emPAI) as an average over the member proteins in the cluster.