title: Mapping protein interactions by combining antibody affinity maturation and mass spectrometry.
abstract: Mapping protein interactions by immunoprecipitation is limited by the availability of antibodies recognizing available native epitopes within protein complexes with sufficient affinity. Here we demonstrate a scalable approach for generation of such antibodies using phage display and affinity maturation. We combined antibody variable heavy (V(H)) genes from target-specific clones (recognizing Src homology 2 (SH2) domains of LYN, VAV1, NCK1, ZAP70, PTPN11, CRK, LCK, and SHC1) with a repertoire of 10(8) to 10(9) new variable light (V(L)) genes. Improved binders were isolated by stringent selections from these new "chain-shuffled" libraries. We also developed a predictive 96-well immunocapture screen and found that only 12% of antibodies had sufficient affinity/epitope availability to capture endogenous target from lysates. Using antibodies of different affinities to the same epitope, we show that affinity improvement was a key determinant for success and identified a clear affinity threshold value (60 nM for SHC1) that must be breached for success in immunoprecipitation. By combining affinity capture using matured antibodies to SHC1 with mass spectrometry, we identified seven known binding partners and two known SHC1 phosphorylation sites in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells. These results demonstrate that antibodies capable of immunoprecipitation can be generated by chain shuffling, providing a scalable approach to mapping protein-protein interaction networks.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Dyson
authors.forename: Yong
authors.initials: Y
authors.lastname: Zheng
authors.forename: Cunjie
authors.initials: C
authors.lastname: Zhang
authors.forename: Karen
authors.initials: K
authors.lastname: Colwill
authors.forename: Kritika
authors.initials: K
authors.lastname: Pershad
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
authors.forename: John
authors.initials: J
authors.lastname: McCafferty
mesh_terms: D000915:Antibody Affinity / Q000276:immunology*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D019151:Peptide Library; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D057127:Single-Chain Antibodies / Q000276:immunology; D018909:src Homology Domains
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D066246:ErbB Receptors