Development of a PNGase Rc column for online deglycosylation of complex glycoproteins during HDX-MS

Lambert, T. O. T.; Gramlich, M.; Stutzke, L. S.; Smith, L.; Deng, D.; Kaiser, P. D.; Rothbauer, U.; Benesch, J. L.; Wagner, C.; Koenig, M.; Pompach, P.; Novak, P.; Zeck, A.; Rand, K. D.

doi:10.1101/2023.07.28.550801

bioRxiv · 10.1101/2023.07.28.550801

Development of a PNGase Rc column for online deglycosylation of complex glycoproteins during HDX-MS

Lambert, T. O. T.·Gramlich, M.·Stutzke, L. S.·Smith, L.·Deng, D.·Kaiser, P. D.·Rothbauer, U.·Benesch, J. L.·Wagner, C.·Koenig, M.·Pompach, P.·Novak, P.·Zeck, A.·Rand, K. D.

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Abstract

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.

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Lambert, T. O. T., Gramlich, M., Stutzke, L. S., Smith, L., Deng, D., Kaiser, P. D., Rothbauer, U., Benesch, J. L., Wagner, C., Koenig, M., Pompach, P., Novak, P., Zeck, A., Rand, K. D.. 2023-07-28. Development of a PNGase Rc column for online deglycosylation of complex glycoproteins during HDX-MS. https://doi.org/10.1101/2023.07.28.550801

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