Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS

• Published in: Journal of the American Society for Mass Spectrometry 2023-11, Vol.34 (11), p.2556-2566
• Main Authors: Lambert, Thomas, Gramlich, Marius, Stutzke, Luisa, Smith, Luke, Deng, Dingyu, Kaiser, Philipp D., Rothbauer, Ulrich, Benesch, Justin L. P., Wagner, Cornelia, Koenig, Maximiliane, Pompach, Petr, Novak, Petr, Zeck, Anne, Rand, Kasper D.
• Format: Article
• Language: English
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1021/jasms.3c00268

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.