Mass spectrometry characterization for chemoenzymatic glycoprotein synthesis.

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TitleMass spectrometry characterization for chemoenzymatic glycoprotein synthesis.
Publication TypeJournal Article
Year of Publication2011
AuthorsSchiel, JE, Lowenthal, MS, Phinney, KW
JournalJ Mass Spectrom
Volume46
Issue7
Pagination649-57
Date Published2011 Jul
ISSN1096-9888
KeywordsAnimals, Cattle, Glycoproteins, Glycosylation, Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase, Mass Spectrometry, Oxazoles, Peptide Fragments, Polysaccharides, Ribonucleases, Trypsin
Abstract

The current project describes the chemoenzymatic modification of bovine ribonuclease B (RNase B) to contain a single glycosylation site with a known glycan. A reactive disaccharide oxazoline derivative was synthesized and stereospecifically added to deglycosylated RNase B 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-MS(n) using four sequential rounds of CID was used to verify that the original glycosylation site had 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. The combined use of chemoenzymatic synthesis and mass spectrometry-based characterization shows promise for the development of homogeneous glycoprotein reference materials. A well-defined glycoprotein standard containing a single glycan of known composition, linkage and stereochemistry would be of great value for the comparison and evaluation of glycoprotein analysis techniques.

DOI10.1002/jms.1934
Alternate JournalJ Mass Spectrom
PubMed ID21706674