How many interactions are there?

~6,200 genes

~6,200 proteins x 2-10 interactions/protein

~12,000 - 62,000 interactions

Yeast

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

Diploid yeastprobed with

DNA-binding domain-Pcf11 bait

fusion protein

Haploid yeastcells expressing

activation domain-prey fusion proteins

High-throughput yeast two-hybrid

protein 1protein 2protein 3protein 4protein 5protein 6

BaitTag

Affinitycolumn

SDS-page

Trypsin digest, identify peptides bymass spectrometry

High-throughput complex mapping by mass spectrometry

493 bait proteins

3617 “interactions”

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

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

Scan with laser

mRNA

fluorescentdyes

cells

genes

experiments

expression levels

Correlation coefficient between expression vectors

True interactionsRandomProteinPairs

Estimating interaction assay accuracy byassessing mRNA coexpression of partners

Estimating interaction assay accuracy byassessing mRNA coexpression of partners

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

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

Quality of the observed protein-protein interactions

Estimating accuracy with a well-determined reference set of interactions

Are interactions between proteins with similar subcellular localization?

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