How many interactions are there? ~6,200 genes ~6,200 proteins x 2-10 interactions/protein ~12,000 -...
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How many interactions are there?
~6,200 genes
~6,200 proteins x 2-10 interactions/protein
~12,000 - 62,000 interactions
Yeast
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Bait
DBD
High-throughput yeast two-hybrid
Bait Prey
DNA bindingdomain
Transcriptionactivation
domain
+
operator orupstream activating
sequence
Reporter gene
transcription
Core transcriptionmachinery
DBD Act
Prey
Act
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Diploid yeastprobed with
DNA-binding domain-Pcf11 bait
fusion protein
Haploid yeastcells expressing
activation domain-prey fusion proteins
High-throughput yeast two-hybrid
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protein 1protein 2protein 3protein 4protein 5protein 6
BaitTag
Affinitycolumn
SDS-page
Trypsin digest, identify peptides bymass spectrometry
High-throughput complex mapping by mass spectrometry
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493 bait proteins
3617 “interactions”
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BaitTag2
Affinitycolumn1
A variant: tandem affinity purification (TAP)
Tag1
Affinitycolumn1
+ protease
Affinitycolumn2
protein 1protein 2protein 3protein 4protein 5protein 6
SDS-page
Trypsin digest, identify peptides bymass spectrometry
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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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Scan with laser
mRNA
fluorescentdyes
cells
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genes
experiments
expression levels
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Correlation coefficient between expression vectors
True interactionsRandomProteinPairs
Estimating interaction assay accuracy byassessing mRNA coexpression of partners
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Estimating interaction assay accuracy byassessing mRNA coexpression of partners
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At least 1 small-scale expmt>1 independent experimentParalogs also interact
Increasing # ofInteraction Sequence
Tags
>1 independent expmt
>2 independent expmtGenome-wide
yeast two-hybrid
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A quantitative measure of pathway reconstruction
Cell cycleMAPK signaling pathway
Swi4 Cdc27Cell cycle
Ubiquitin-mediated proteolysis
Systematically test every pair of characterized proteins
Jaccard coefficient = # pathways in common / # total pathways
Pathwaysof A
Pathwaysof B
pw1 pw2
U
U
pw1 pw2n
pairs
1n<pathway similarity> =
pw1 pw2
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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
Authors Method # interactions Mrowka Deane vonMering MarcotteIto 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%
>1 independent experiment ~2000 85% ~30-40% ~67%>2 independent experiments 1167 88% ~60-70% ~73%
Co-expression Test set Pathways
Estimated True Positive Ratevia