RESUMO
Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.
Assuntos
Mapeamento de Epitopos , Receptor ErbB-2 , Trastuzumab , Humanos , Mapeamento de Epitopos/métodos , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Trastuzumab/química , Alquilação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Halogenação , Pegadas de Proteínas/métodos , Complexo Antígeno-Anticorpo/químicaRESUMO
A series of fluoroalkylated cyclic λ3 -iodanes and their hydrochloride salts was prepared and used in a combination with sodium ascorbate in buffer or aqueous methanol mixtures for radical fluoroalkylation of a range of substituted indoles, pyrroles, tryptophan or its derivatives, and Trp residues in peptides. As demonstrated on several peptides, the aromatic amino acid residues of Trp, Tyr, Phe, and His are targeted with high selectivity to Trp. The functionalization method is biocompatible, mild, rapid, and transition-metal-free. The proteins myoglobin, ubiquitin, and human carbonic anhydraseâ I were also successfully functionalized.