ArticleAnalytical and bioanalytical chemistry2026
An ionic liquid-functionalized styrene-maleic anhydride copolymer brush stationary phase coupled with offline two-dimensional LC-MS/MS for comprehensive exosomal phospholipid profiling in breast cancer.
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Abstract
Comprehensive characterization of exosomal phospholipids is essential for understanding cancer-associated metabolic alterations and discovering potential biomarkers, but remains challenging due to their extensive structural diversity and the limited selectivity and coverage of conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches. In this study, an ionic liquid-functionalized styrene-maleic anhydride copolymer brush stationary phase (Sil-BHHICl) was developed and integrated into an offline two-dimensional (2D) LC-MS/MS platform for comprehensive exosomal phospholipid profiling. The Sil-BHHICl stationary phase exhibited a hydrophilic interaction/reversed-phase mixed-mode retention mechanism and provided enhanced selectivity toward phospholipids, enabling efficient separation at both the phospholipid-class and molecular-species levels. The developed analytical platform enabled comprehensive profiling of plasma-derived exosomal phospholipids, resulting in the identification of 274 molecular species belonging to 10 phospholipid classes. Lipidomic analysis of plasma exosomes from breast cancer patients and healthy controls revealed distinct phospholipid alterations associated with breast cancer and identified five candidate biomarkers, among which LysoPC-O (16:0) exhibited the strongest discriminatory ability. Pathway enrichment analysis further indicated that the altered phospholipids were predominantly associated with glycerophospholipid metabolism, suggesting extensive lipid metabolic remodeling in breast cancer. Overall, this study establishes a mixed-mode chromatographic platform for high-coverage exosomal phospholipid profiling and demonstrates its potential for lipidomics-based biomarker discovery and investigation of disease-associated metabolic alterations.
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