How many interactions are there?

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How many interactions are there?
Yeast
6,200 genes
6,200 proteins x 2-10 interactions/protein
12,000 - 62,000 interactions
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High-throughput yeast two-hybrid

DBD
Bait
Prey
Act
DNA binding domain
Transcription activation domain
Core transcription machinery
transcription
operator or upstream activating sequence
Reporter gene
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High-throughput yeast two-hybrid
Haploid yeast cells expressing activation
domain- prey fusion proteins
Diploid yeast probed with DNA-binding
domain- Pcf11 bait fusion protein
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High-throughput complex mapping by mass
spectrometry
Tag
Bait
Affinity column
protein 1
protein 2
protein 3
SDS- page
Trypsin digest, identify peptides by mass
spectrometry
protein 4
protein 5
protein 6
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493 bait proteins 3617 interactions
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A variant tandem affinity purification (TAP)
Tag2
Tag1
Bait
Affinity column2
protein 1
protein 2
Affinity column1
protein 3
SDS- page
protein 4
protein 5
protease
protein 6
Trypsin digest, identify peptides by mass
spectrometry
Affinity column1
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But just how accurate are these high-throughput
screens?
Four tests 1. Comparison of mRNA co-expression
of interacting partners 2. Comparison of
interactions to a test set 3. Comparison of
functions of predicted interaction partners 4.
Comparison of subcellular localization of
interaction partners
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cells
mRNA
fluorescent dyes
Scan with laser
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experiments
expression levels
genes
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Estimating interaction assay accuracy
by assessing mRNA coexpression of partners
Random Protein Pairs
True interactions
Correlation coefficient between expression vectors
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Estimating interaction assay accuracy
by assessing mRNA coexpression of partners
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gt1 independent expmt
gt2 independent expmt
Genome-wide yeast two-hybrid
At least 1 small-scale expmt gt1 independent
experiment Paralogs also interact
Increasing of Interaction Sequence Tags
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A quantitative measure of pathway reconstruction
Swi4
Cdc27
Cell cycle Ubiquitin-mediated proteolysis
Cell cycle MAPK signaling pathway
Pathways of A
Pathways of B
pw1
pw2
Jaccard coefficient pathways in common /
total pathways
S
ltpathway similaritygt
n pairs
Systematically test every pair of characterized
proteins
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Quality of the observed protein-protein
interactions
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Estimating accuracy with a well-determined
reference set of interactions
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Are interactions between proteins with similar
subcellular localization?
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Summary of tests
Estimated True Positive Rate via
Co-expression Test set Pathways
Authors Method
interactions Mrowka Deane vonMering Marcotte Ito
et al. Y2H 4081 9 22 6-10 14 Ho
et al. MS 3617 10 1-3 Gavin et
al. MS 1440 85 10 Uetz et al. Y2H
957 53 50 44 Tong et al. synthetic
lethal 295 20 gt1 independent
experiment 2000 85 30-40 67 gt2
independent experiments 1167
88 60-70 73