Mapping protein interactions by combining antibody affinity maturation and mass spectrometry

Mapping protein interactions by immunoprecipitationis 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 displayand affinity maturation. We combined antibody variable heavy (VH) genes from target-specific clones (recognizing Src homology 2 ( SH2) domainsof LYN, VAV1, NCK1, ZAP70, PTPN11, CRK, LCK, and SHC1) with a repertoire of 108 to 109 new variable light (VL) 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 affinitiesto the same epitope, we show that affinityimprovement was a key determinant for success and identified a clear affinitythreshold value (60 nM for SHC1) that must be breached for success in immunoprecipitation. By combining affinitycapture using matured antibodies to SHC1with mass spectrometry, we identified seven known binding partners and two known SHC1phosphorylation sites in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells. These results demonstrate that antibodies capable of immunoprecipitationcan be generated by chain shuffling, providing a scalable approach to mapping protein– protein interaction networks.