Dissecting the glycoprofile of the human cytomegalovirus protein gB by comprehensive mass spectrometry analysis

Glycosylation is the most critical post-translational modification occurring on proteins and its specific pattern depends both on the cells used for their production, such as mammalian cells, insect cells and yeast, and the expression conditions (e.g., mannosidase inhibitors, gene deletion). The aim of this study is to characterize the glycans of recombinant glycoprotein B (gB) from Human Cytomegalovirus (HCMV), expressed in CHO cells, by using different hyphenated mass spectrometry techniques in order to enrich the knowledge about glycosylation pattern changes in the protein production process and to develop versatile analytical platforms for the rapid analysis of protein glycosylation. The settled workflows focus on two main classes of analytes originating from the glycoprotein studied: glycopeptides and glycans. The analysis of the first category of molecules relies on a bottom-up proteomic approach in which the glycosites and the correspondent glycan heterogeneity and occupancy have been investigated by using high resolution tandem mass spectrometry; the analysis of the second class of molecules occurs by glycan shaving followed by fluorescence-liquid chromatography and mass-spectrometry characterization. These analyzes have been performed on the same glycoprotein expressed in a different culture media to demonstrate the capability of these analytical methods to monitor in detail oligosaccharide pattern changes. Finally, the impact of changes in glycosylation pattern on antigen immunogenicity has been assessed, showing that the presence of only high mannose oligosaccharides elicits higher titer of neutralizing antibodies.