title: Fast photochemical oxidation of proteins (FPOP) maps the epitope of EGFR binding to adnectin.
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 ((10)Fn3). 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.
authors.affiliation: Center for Biomedical and Bioorganic Mass Spectrometry, Department of Chemistry, Washington University in St. Louis, St. Louis, MO, 63130-4899, USA.
authors.forename: Yuetian
authors.initials: Y
authors.lastname: Yan
authors.forename: Guodong
authors.initials: G
authors.lastname: Chen
authors.forename: Hui
authors.initials: H
authors.lastname: Wei
authors.forename: Richard Y-C
authors.initials: RY
authors.lastname: Huang
authors.forename: Jingjie
authors.initials: J
authors.lastname: Mo
authors.forename: Don L
authors.initials: DL
authors.lastname: Rempel
authors.forename: Adrienne A
authors.initials: AA
authors.lastname: Tymiak
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
mesh_terms: D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D000939:Epitopes; D005353:Fibronectins; C512478:EGFR protein, human; D066246:ErbB Receptors