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

“Intact mass and peptide mapping approaches to characterize lipoprotein species of fHbp. Long Z(1), Li M(2), Zhu X(1), Luo X(3), Wang B(4). Author information: (1)ThermoFisher Scientific Corporation, Beijing 100080, China. (2)National Institutes for Food and Drug Control, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, State Key Laboratory of Drug Regulatory Science, Beijing 102629, China. (3)State Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. Electronic address: xluo@ipe.ac.cn. (4)National Institutes for Food and Drug Control, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, State Key Laboratory of Drug Regulatory Science, Beijing 102629, China. Electronic address: binwang@nifdc.org.cn. 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. Copyright © 2026. Published by Elsevier B.V. Conflict of interest statement: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.”