Intact mass and peptide mapping approaches to characterize lipoprotein species of fHbp

Lipoproteins are promising vaccine candidates due to their bioactivity and self-adjuvanting properties, but complex lipid modifications essential for immunomodulatory function pose analytical challenges. Previous approaches combine peptide mass data from liquid chromatography-mass spectrometry (LC-MS) with lipid abundance data from gas chromatography-mass spectrometry, providing only indirect characterization. In this study, we developed a streamlined LC-MS strategy that combined high-resolution mass spectrometry with electron-transfer/higher-energy collision dissociation (EThcD) to enable direct and accurate characterization of lipid modifications at both intact protein and peptide levels. By integrating intact protein and peptide-level analysis, this approach provided detailed structural information on lipoprotein modifications. Using Neisseria meningitidis serogroup B factor H binding protein (fHbp) as a model, thirteen distinct lipid modifications were identified and relatively quantified, including seven tri-acylated and six di-acylated forms. The major fatty acid components (C16:0, C16:0, and C17:1) accounted for approximately 50% of the total fatty acids. The method also revealed differences in lipid distributions between manufacturers and changes during storage, with tri-acylated forms decreasing in favor of di-acylated species. This approach provides a robust and straightforward alternative for characterization, quality control, and stability assessment of lipoprotein-based vaccines and can also support comparability studies following manufacturing changes.