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Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

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Journal of The American Society for Mass Spectrometry

Abstract

Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein–protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.

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Acknowledgments

This research was supported by the NIH, NIGMS P41 grant (P41GM103422) to M.L.G. The authors thank Dr. Reb Russell, Dr. Sharon Cload, and Dr. Bruce Car from Bristol-Myers Squibb for their support of this project.

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Authors and Affiliations

  1. Center for Biomedical and Bioorganic Mass Spectrometry, Department of Chemistry, Washington University in St. Louis, St. Louis, MO, 63130-4899, USA

    Yuetian Yan, Don L. Rempel & Michael L. Gross

  2. Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb, Princeton, NJ, 08540-4715, USA

    Guodong Chen, Hui Wei, Richard Y.-C. Huang, Jingjie Mo & Adrienne A. Tymiak

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  1. Yuetian Yan
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  2. Guodong Chen
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Correspondence to Michael L. Gross.

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Yan, Y., Chen, G., Wei, H. et al. Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin. J. Am. Soc. Mass Spectrom. 25, 2084–2092 (2014). https://doi.org/10.1007/s13361-014-0993-x

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