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Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS
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SYSNO ASEP 0581603 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS Author(s) Lambert, T. (DK)
Gramlich, M. (DE)
Stutzke, L. (DK)
Smith, L. (GB)
Deng, D. (DK)
Kaiser, Philipp D. (DE)
Rothbauer, U. (DE)
Benesch, J. L. P. (GB)
Wagner, C. (DE)
Koenig, M. (DE)
Pompach, Petr (BTO-N)
Novák, Petr (MBU-M) RID, ORCID
Zeck, A. (DE)
Rand, K. D. (DK)Source Title Journal of the American Society for Mass Spectrometry. - : American Chemical Society - ISSN 1044-0305
Roč. 34, č. 11 (2023), s. 2556-2566Number of pages 11 s. Language eng - English Country US - United States Keywords hydrogen/deuterium exchange ; resolution ; proteins OECD category Biochemistry and molecular biology R&D Projects LM2023042 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) EF18_046/0015974 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Research Infrastructure CIISB III - 90242 - Masarykova univerzita / Středoevropský technologický institut Method of publishing Open access Institutional support MBU-M - RVO:61388971 ; BTO-N - RVO:86652036 UT WOS 001074666000001 EID SCOPUS 85175202999 DOI 10.1021/jasms.3c00268 Annotation 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. Workplace Institute of Microbiology Contact Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Year of Publishing 2024 Electronic address https://pubs.acs.org/doi/10.1021/jasms.3c00268
Number of the records: 1