1. Protein interactions: main methods for detection (all organisms) Two-hybrid8,446 (Co-)Immunoprecipitation567 Interaction adhesion assay225 In vitro binding138 Affinity Column120 Copurification85 Immunoblotting83 Other Biophysical73 All interactions10,432 Source: The Database of Interacting Proteins (DIP)

2. Protein interactions: top 8 organisms Yeast (Saccharomyces cerevisiae)7,881 Helicobacter pylori1,418 Man (Homo sapiens )595 Worm (Caenorhabditis elegans)~500 Mouse (Mus musculus )130 Fly (Drosophila melanogaster)81 Escherichia coli56 Rat (Rattus norvegicus )48 All interactions10,432 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. Mass spectrometry Protein chips (arrays) Biochemical proteomics Extented by recent contributions from 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

5. Two-hybrid-Array (rich media) HIS3 DBD B AD ORF HIS3 DBD B HIS3 DBD B ORF AD ORF AD

6. HIS3 DBD B AD ORF HIS3 DBD B HIS3 DBD B ORF AD ORF AD Two-hybrid-Array (-His media) bait: PCF11

7. Details in Cagney & Uetz, Methods Enzymol. 328: 3 (2000); Current Protocols in Protein Science 19.6 (2001) rich- Histidine Insert movie 1 here Insert movie 2 here

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 interactionsMaximum: 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. Cdc53 Cep3 Eft1 Hrt1 Rav1 Rav2 Rcy1 (F) YBR280 (F) YJL149 (F) YMR258C (F) MS1 Seol et al. 2001 Nature Cell Biol. 3: 384 MS3 (Aebersold et al., pers. comm.) Grr1 (F) YLR097C (F) Bdf1 Cdc4 Ctf13 (F) Met30 (F) Rub1 Sgt1 YLR224W (F) YLR352W (F) YLR368W (F) Two-Hybrid Uetz et al. 2000 Nature 403: 623 Skp1

12. A B C D E F G H I J K L M protein pulled down with epitope-tagged protein B protein pulled down with epitope-tagged protein E two-hybrid interactions Two-hybrid…vs. mass spec

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 1Protein 2 Protein pairs Domain information from the Interpro database (Apweiler et al., Nucl. Acids Res. 29; 37 [2001]) Conclusion:

15. Predicting Protein Interaction Domains Based on Sprinzak & Margalit, J. Mol. Biol. 311: 681 [2001] domain combinations 4 log-odds score: log 2 (-------) observed frequency of pair frequencies of domains P ij PiPjPiPj

16. Summary (sort of…) Protein interactions in the Database of Interacting Proteins (DIP) All interactions10,432 PDB entries~1900 one domain mapped252 both domains mapped175 Dissociation constant available26 Source: The Database of Interacting Proteins (DIP) as of late 2001