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

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.