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

0581603 - MBÚ 2024 RIV US eng J - Journal Article
Lambert, T. - Gramlich, M. - Stutzke, L. - Smith, L. - Deng, D. - Kaiser, Philipp D. - Rothbauer, U. - Benesch, J. L. P. - Wagner, C. - Koenig, M. - Pompach, Petr - Novák, Petr - Zeck, A. - Rand, K. D.
Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS.
Journal of the American Society for Mass Spectrometry. Roč. 34, č. 11 (2023), s. 2556-2566. ISSN 1044-0305. E-ISSN 1879-1123
R&D Projects: GA MŠMT LM2023042; GA MŠMT(CZ) EF18_046/0015974
Research Infrastructure: CIISB III - 90242
Institutional support: RVO:61388971 ; RVO:86652036
Keywords : hydrogen/deuterium exchange * resolution * proteins
OECD category: Biochemistry and molecular biology
Impact factor: 3.2, year: 2022
Method of publishing: Open access
https://pubs.acs.org/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.
Permanent Link: https://hdl.handle.net/11104/0349703