Mapping protein interactions by combining antibody affinity maturation and mass spectrometry.
Mapping protein interactions by combining antibody affinity maturation and mass spectrometry.
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DOI：
10.1016/j.ab.2011.05.005
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发表时间：
2011-10-01
影响因子：
2.9
通讯作者：
McCafferty J
中科院分区：
文献类型：
--
作者：
Dyson MR;Zheng Y;Zhang C;Colwill K;Pershad K;Kay BK;Pawson T;McCafferty J
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.
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DOI：
10.1093/jnci/53.3.661
发表时间：
1974-01-01
期刊：
JOURNAL OF THE NATIONAL CANCER INSTITUTE
影响因子：
--
作者：
CAILLEAU, R;YOUNG, R;REEVES, WJ
通讯作者：
REEVES, WJ
影响因子：
6.8
作者：
Gai, S. Annie;Wittrup, K. Dane
通讯作者：
Wittrup, K. Dane
影响因子：
56.9
作者：
Hardy, W. Rod;Li, Lingying;Pawson, Tony
通讯作者：
Pawson, Tony
DOI：
10.1073/pnas.88.24.11120
发表时间：
1991-12-01
影响因子：
11.1
作者：
KANG, AS;JONES, TM;BURTON, DR
通讯作者：
BURTON, DR
影响因子：
7
作者：
Huang, Haiming;Li, Lei;Li, Shawn S. -C.
通讯作者：
Li, Shawn S. -C.