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)

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)

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, /~4,700 ========== ~2, ,200

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, /~4,700 ========== ~2, ,200

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

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

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

Analysis of Yeast Protein Complexes by Mass spec Gavin et al. (2002) Nature 415: epitope-tagged proteins (include 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

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

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

Cdc53 Cep3 Eft1 Hrt1 Rav1 Rav2 Rcy1 (F) YBR280 (F) YJL149 (F) YMR258C (F) MS1 Seol et al 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 Nature 403: 623 Skp1

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

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

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:

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

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