Date Published: October 01, 2011
Publisher: Academic Press
Author(s): Michael R. Dyson, Yong Zheng, Cunjie Zhang, Karen Colwill, Kritika Pershad, Brian K. Kay, Tony Pawson, John McCafferty.
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 (VH) 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 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 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.
The combination of antibody affinity capture coupled with MS provides a powerful platform for mapping protein–protein interactions. The widespread use of this technique, however, is limited by the availability of suitable antibodies. Understanding the requirements for immunoprecipitation and increasing the availability of such reagents would be of huge advantage in mapping protein interaction networks, particularly if this could be applied in a high-throughput manner. Several factors, including the properties of the targeted epitope, determine the utility of individual antibodies for immunoprecipitation. In techniques such as Western blotting and immunohistochemistry, the antibody must recognize an epitope, typically linear, that survives the denaturation conditions used. In contrast, immunoprecipitation requires antibodies that recognize the native proteins. Depending on the supramolecular architecture of the protein complex, some epitopes will not be accessible for antibody capture because they will be occluded by other interacting proteins within the complex. Finally, affinity is also important because the method requires capture of endogenous protein present at low levels in cell lysates and also retention during the extensive wash steps employed after the complex has been captured.
We have presented a scalable method for antibody affinity maturation, based on chain shuffling, to generate improved phage antibodies to a set of eight SH2 domain proteins. For each antigen, we matured oligoclonal mixes of clones that previously had been shown to have exquisite specificity for the target antigen, and we demonstrated that it is possible to rapidly and simultaneously affinity mature multiple specific antibodies to multiple different antigens. Using an affinity capture screen, we found that only a minority of affinity matured scFvs were successful in the capture of target antigens from lysates, and this is likely due to a combined requirement for affinity and the need to recognize available epitopes on native protein. Affinity improvements were a key determinant in the success of immunoprecipitation demonstrated using panels of antibodies with differing affinities to the same epitope. This systematic approach not only illustrated the importance of affinity but also identified a relatively sharp cutoff distinguishing between success and failure. Finally, we validated this approach by combining affinity capture using matured antibodies with MS to identify seven binding partners of SHC1 in EGF-stimulated cells. This approach, combined with MS, will help to advance our knowledge and understanding of protein interactions occurring during cell signaling.