FDR Thresholding

1
FDR Thresholding

• Caleb J. Emmons

2
What is FDR?
If decoy proteins are present
decoy proteins identified
Protein FDR
target proteins identified
3
The FDR Browser
4
How does FDR Thresholding work?
5
4
Minimum number of peptides
3
2
1
The FDR Landscape
5
How does FDR Thresholding work?
The FDR Landscape
6
Some Fine Points
Confusing!
7
Protein Clustering

• Poster 509, Tuesday 1030-100
• Informatics Quantification/Validation
• Caleb J. Emmons

8
What is a Cluster?
9
Total Peptide Evidence
Protein PEtot
K1C10 1481
K1C14 1061
K1C16 852
K1C17 503
10
Joint Peptide Evidence
PEjoint K1C10 K1C14 K1C16
K1C14 184
K1C16 184 661
K1C17 84 375 175
11
Cluster Formation
Directly similar
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
Clusters
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.
12
Cluster Formation
Protein PEtot
K1C10 1481
K1C14 1061
K1C16 852
K1C17 503
PEjoint K1C10 K1C14 K1C16
K1C14 184
K1C16 184 661
K1C17 84 375 175
A B ? K1C10 K1C14 K1C16
K1C14 no
K1C16 no yes
K1C17 no yes no
13
Peptide-Protein Weights
A B C
 
14
Spectrum Counting
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

Protein A 3 2 3 1
Protein B 4 2 4 2
Protein C 4 2 3 1
Cluster of BC 6 5 5 4
Unique Spectra
Total Spectra
Exclusive Spectra
Exclusive Unique Peptides
B
A
C
15
Quantitative Values
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. Normalize
d 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.