ArticleAnalytical biochemistry2011
Mapping protein interactions by combining antibody affinity maturation and mass spectrometry.
Article in Analytical biochemistry, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 23 citations in OpenAlex.
- Generation and validation of recombinant antibodies to study human aminoacyl-tRNA synthetases.The Journal of biological chemistry · 2020Article
- Article
- Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers.Oncotarget · 2017Article
- Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain.Scientific reports · 2017Article
- Article
- Applications for an engineered Protein-G variant with a pH controllable affinity to antibody fragments.Journal of immunological methods · 2014Article
- Review
- Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells.Proceedings of the National Academy of Sciences of the United States of America · 2013Article
- Generation of high-performance binding proteins for peptide motifs by affinity clamping.Methods in enzymology · 2013Article
- Generation of anti-Notch antibodies and their application in blocking Notch signalling in neural stem cells.Methods (San Diego, Calif.) · 2012Article
- Combining different design strategies for rational affinity maturation of the MICA-NKG2D interface.Protein science : a publication of the Protein Society · 2012Article
- Rapid identification of monospecific monoclonal antibodies using a human proteome microarray.Molecular & cellular proteomics : MCP · 2012Article
- Detection of biomarkers using recombinant antibodies coupled to nanostructured platforms.Nano reviews · 2012Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
8 authors at 4 institutions in 3 countries.
Funding
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.