| Autor: |
Lambert T; Department of Pharmacy, University of Copenhagen, 2100 Copenhagen, Denmark., Gramlich M; NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany., Stutzke L; Department of Pharmacy, University of Copenhagen, 2100 Copenhagen, Denmark., Smith L; Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, OX1 3QZ Oxford, England., Deng D; Department of Pharmacy, University of Copenhagen, 2100 Copenhagen, Denmark., Kaiser PD; NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany., Rothbauer U; NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany.; Pharmaceutical Biotechnology, Eberhard Karls University, 72074 Tübingen, Germany., Benesch JLP; Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, OX1 3QZ Oxford, England., Wagner C; Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, 82377 Penzberg, Germany., Koenig M; Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, 82377 Penzberg, Germany., Pompach P; BioCev, Institute of Biotechnology of the CAS, 252 50 Prumyslova, Czech Republic., Novak P; BioCeV, Institute of Microbiology of the CAS, 142 20 Prumyslova, Czech Republic., Zeck A; NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany., Rand KD; Department of Pharmacy, University of Copenhagen, 2100 Copenhagen, Denmark. |
| Abstrakt: |
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. |
