Protein interactions: main methods for detection

1
Protein interactions main methods for detection
(all
organisms) Two-hybrid 8,446 (Co-)Immunoprecipita
tion 567 Interaction adhesion assay 225 In vitro
binding 138 Affinity Column 120 Copurification 85
Immunoblotting 83 Other Biophysical 73 All
interactions 10,432
Source The Database of Interacting Proteins (DIP)
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Protein interactions top 8 organisms Yeast
(Saccharomyces cerevisiae) 7,881 Helicobacter
pylori 1,418 Man (Homo sapiens ) 595 Worm
(Caenorhabditis elegans) 500 Mouse (Mus musculus
) 130 Fly (Drosophila melanogaster) 81 Escherichia
coli 56 Rat (Rattus norvegicus ) 48 All
interactions 10,432
Source The Database of Interacting Proteins (DIP)
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Up to 6,200 Protein interactions reported in 5
papers Fromont-Racine et al.1997, 2000
300 Uetz et al./Drees et al. 2000,
2001 1,200 Ito et al. 2000, 2001
841/4,700 2,500
- 6,200
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Up to 6,200 Protein interactions reported in 5
papers Fromont-Racine et al.1997, 2000
300 Uetz et al./Drees et al. 2000,
2001 1,200 Ito et al. 2000, 2001
841/4,700 2,500
- 6,200
Mass spectrometry Protein chips (arrays)
Biochemical proteomics
Extented by recent contributions from
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Two-hybrid-Array (rich media)
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Two-hybrid-Array (-His media)
bait PCF11
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Insert movie 1 here
Insert movie 2 here
rich
- Histidine
Details in Cagney Uetz, Methods Enzymol. 328 3
(2000) Current Protocols in Protein Science 19.6
(2001)
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Analysis of Yeast Protein Complexes by Mass spec
Gavin et al. (2002) Nature 415 141-147 1739
epitope-tagged proteins (include. 1143 human
orthologues) 1167 clones expressed the tagged
protein 589 successful purifications 232
distinct complexes identified 134 new
complexes 16,830 protein bands identified
(corresponding to 1440 proteins) Note
The probability to detect the same protein in a
complex twice is 70 Conclusion there are
about 700 complexes in yeast
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Analysis of Yeast Protein Complexes by Mass spec
How many protein interactions are there based on
mass spec data? On average 12 proteins per
complex,
Minimum 11 interactions
Maximum 55 interactions
? 8,000 - 38,500 interactions total
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Analysis of Yeast Protein Complexes by Mass spec
Our two-hybrid array (!) screens for
comparison 650 baits screened against
whole-genome array 330 baits yielded 1200
reproducible interactions of those
1200 only 100 showed up in complexes 76
proteins used as both baits/entry points by
Gavin et al. yielded 227 interactions in our
two-hybrid screens (i.e. 2.9 on average) 76
purifications complexes would contain 900
proteins with x interactions
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MS3 (Aebersold et al., pers. comm.)
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Two-hybridvs. mass spec
K
G
J
A
E
H
C
F
L
B
D
M
I
two-hybrid interactions
protein pulled down
protein pulled down
with epitope-tagged
with epitope-tagged
protein B
protein E
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Limitations of current network graphs -
static - no localization data - no or limited
integration with other compounds - no or limited
data on experimental conditions - no or limited
data on expression levels
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Predicting Protein Interaction Domains
Based on Sprinzak Margalit, J. Mol. Biol. 311
681 2001
Protein 1
Protein 2
Protein pairs
Conclusion
Domain information from the Interpro database
(Apweiler et al., Nucl. Acids Res. 29 37 2001)
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Predicting Protein Interaction Domains
Based on Sprinzak Margalit, J. Mol. Biol. 311
681 2001
domain combinations
log-odds score log2 (-------)
observed frequency of pair
Pij
frequenciesof domains
PiPj
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Summary (sort of) Protein interactions in the
Database of Interacting Proteins (DIP) All
interactions 10,432 PDB entries 1900 one
domain mapped 252 both domains
mapped 175 Dissociation constant available 26
Source The Database of Interacting Proteins
(DIP) as of late 2001