Mass spectrometric analysis of lipid-linked oligosaccharide heterogeneity in genetically modified E. coli used for glycoconjugate bacterial vaccine production.

Polysaccharide-based glycoconjugate bacterial vaccines can help combat multidrug-resistant pathogens by harnessing the immune system to recognize and neutralize bacteria effectively. Bacterial cells can be engineered to link O-antigen polysaccharides (O-PSs) to a carrier protein, producing a potential bacterial vaccine, e.g., against extraintestinal pathogenic Escherichia coli (ExPEC). Lipid-linked oligosaccharides (LLOs) play a key role in the bioconjugation process, and determining their heterogeneity is essential to evaluate the variability among clinical isolates and the success of the bioengineering process. Mass spectrometry (MS) techniques can provide unique structural insights, but applications have typically been limited to short LLOs or hydrolyzed LLO fragments. Here, we used ultrahigh-resolution MALDI MS to analyze LLOs from an engineered E. coli strain expressing E. coli O2 O-PSs. Two methods were evaluated: (1) analysis of LLOs after extraction and purification, and (2) analysis of hydrolyzed O-PS fragments obtained by mild acidic hydrolysis of bacterial biomass. Negative and positive ion mode measurements revealed LLO structural polydispersity and heterogeneity, while MS/MS confirmed RU monosaccharide composition and structural consistency with glycoconjugate-derived O-antigens. The hydrolysis-based approach also enabled rapid RU profiling with some limitations. This study highlights MALDI FT-ICR MS as an effective tool for analyzing bacterial LLOs and advancing ExPEC-targeted glycoconjugate vaccines. HYPOTHESIS STATEMENT: We hypothesize that ultrahigh-resolution MALDI FT-ICR mass spectrometry can reliably characterize the structural heterogeneity and repeat unit composition of lipid-linked oligosaccharides (LLOs) from engineered E. coli strains, thereby providing critical analytical insights to support the development of glycoconjugate vaccines against extraintestinal pathogenic E. coli (ExPEC).