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.

• MeSH
• Alkylation MeSH
• Protein Footprinting methods   MeSH
• Halogenation MeSH
• Antigen-Antibody Complex chemistry   MeSH
• Humans MeSH
• Epitope Mapping * methods   MeSH
• Antibodies, Monoclonal chemistry   immunology   MeSH
• Receptor, ErbB-2 * chemistry   immunology   MeSH
• Trastuzumab * chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

• MeSH
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase MeSH
• Glycoproteins * analysis   MeSH
• Glycosylation MeSH
• Polysaccharides * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase MeSH
• Glycoproteins * MeSH
• Polysaccharides * MeSH