Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane

Analysis of protein modifications is critical for quality control of therapeutic biologics. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. A method utilizing chemoselective labeling of glycated lysine residues, through rapid conversion of site-specific glycation to stable N-ethylmaleimide derivatives at mildly acidic conditions, identified highly reactive lysine sites of proteins. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase.