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Protein interactions: main methods for detection

Protein interactions: main methods for detection (all organisms) Two-hybrid 8,446 (Co-)Immunoprecipitation 567 Interaction adhesion assay 225 In vitro binding 138 Affinity Column 120 Copurification 85 Immunoblotting 83 Other Biophysical 73

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Protein interactions: main methods for detection

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  1. Protein interactions: main methods for detection (all organisms) Two-hybrid 8,446 (Co-)Immunoprecipitation 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)

  2. 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)

  3. 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

  4. 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 Extented by recent contributions from • Mass spectrometry • Protein chips (arrays) • Biochemical proteomics

  5. O R F H I S 3 H I S 3 A D O R F A D A D B B O R F B D B D D B D D B D H I S 3 Two-hybrid-Array (rich media)

  6. O R F H I S 3 H I S 3 A D O R F A D A D B B O R F B D B D D B D D B D H I S 3 Two-hybrid-Array (-His media) bait: PCF11

  7. 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)

  8. 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

  9. 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

  10. 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

  11. MS1 Seol et al. 2001 Nature Cell Biol. 3: 384 Rav1 Cep3 Cdc53 Rav2 YBR280 (F) Eft1 R c y 1 ( F ) Y J L 1 4 9 ( F ) Y M R 2 5 8 C ( F ) H r t 1 YLR097C (F) Skp1 G r r 1 ( F ) Y L R 3 6 8 W ( F ) B d f 1 Y L R 3 5 2 W ( F ) C d c 4 Y L R 2 2 4 W ( F ) R u b 1 C t f 1 3 ( F ) Two-Hybrid Uetz et al. 2000 Nature 403: 623 S g t 1 M e t 3 0 ( F ) MS3 (Aebersold et al., pers. comm.)

  12. Two-hybrid…vs. 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

  13. 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

  14. 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])

  15. 4 Predicting Protein Interaction Domains Based on Sprinzak & Margalit, J. Mol. Biol. 311: 681 [2001] domain combinations log-odds score: log2 (-------) Pij observed frequency of pair PiPj frequenciesof domains

  16. 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

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