Comprehensive characterization of bacterial glycoconjugate vaccines by liquid chromatography - mass spectrometry
Abstract
Bacterial pathogens can cause a broad range of infections with detrimental effects on health. Vaccine development is essential as multi -drug resistance in bacterial infections is a rising concern. Recombinantly produced proteins carrying O -antigen glycosylation are promising glycoconjugate vaccine candidates to prevent bacterial infections. However, methods for their comprehensive structural characterization are lacking. Here, we present a bottom -up approach for their site -specific characterization, detecting N -glycopeptides by nano reversed -phase liquid chromatography -mass spectrometry (RP -LC -MS). Glycopeptide analyses revealed information on partial site -occupancy and site -specific glycosylation heterogeneity and helped corroborate the polysaccharide structures and their modifications. Bottom -up analysis was complemented by intact glycoprotein analysis using nano RP -LC -MS allowing the fast visualization of the polysaccharide distribution in the intact glycoconjugate. At the glycopeptide level, the model glycoconjugates analyzed showed different repeat unit (RU) distributions that spanned from 1 to 21 RUs attached to each of the different glycosylation sites. Interestingly, the intact glycoprotein analysis displayed a RU distribution ranging from 1 to 28 RUs, showing the predominant species when the different glycopeptide distributions are combined in the intact glycoconjugate. The complete workflow based on LC -MS measurements allows detailed and comprehensive analysis of the glycosylation state of glycoconjugate vaccines.
