Mass Spectrometry Characterization for Chemoenzymatic Glycoprotein Synthesis
John E. Schiel, Mark S. Lowenthal, Karen W. Phinney
Glycoproteins exist as heterogeneous mixtures of glycoforms due to glycosylation at multiple sites with various glycans. The glycosylation pattern has significant implications for clinical diagnostics and biopharmaceutical development because it affects stability, toxicity, and activity. Complete glycoprotein characterization involves numerous multidisciplinary techniques, including many invaluable mass spectrometry-based approaches. A well defined glycoprotein standard containing a single glycan of known composition, linkage, and steroechemistry would be of great value for the comparison and evaluation of glycoprotein analysis techniques. The current project describes the rapid one-pot chemoenzymatic rearrangement of bovine ribonuclease B (RNase B) to contain a single glycosylation site with a known trisaccharide glycan. The procedure involved enzymatic removal of native glycans from the protein, leaving only the penultimate N-acetylglucosamine residue attached. A reactive disaccharide oxazoline derivative was then synthesized and stereospecifically added to the deglycosylated RNase through Endo-β-N-Acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation. Oxazoline formation conditions were optimized using mass spectrometry, and the product verified based on its collision induced dissociation (CID) mass spectrum. Enzymatic removal of native glycans as well as formation of the desired homogeneous product was also monitored using mass spectrometry. LC-MSn using four sequential rounds of CID was used to verify that the original glycosylation site had in fact been reorganized to contain the new glycan. The techniques described herein are not limited to this analyte or glycan, and should be amenable to the synthesis of numerous homogeneous glycoconjugates with judicious choice of enzyme/substrate combinations. These materials may prove useful for increasing our understanding of currently used and innovative glycoprotein analysis techniques.
, Lowenthal, M.
and Phinney, K.
Mass Spectrometry Characterization for Chemoenzymatic Glycoprotein Synthesis, Glycoconjugate Journal, [online], https://doi.org/10.1002/jms.1934
(Accessed March 5, 2024)