13 C-Isotope Tracing Structural Lipidomics for Resolving Phospholipid Metabolism Dynamics in Human Breast Cancer Cells

Phospholipid metabolic homeostasis is critical, yet its dynamic regulation at the structural level remains poorly characterized, particularly from the perspective of metabolic flux analysis. This study presents an RPLC–PB–MS/MS workflow integrating 13 C-glucose isotope tracing and the Paternò–Büchi (PB) reaction for comprehensive investigation of de novo phospholipid synthesis. By precisely mapping 13 C labeling sites, we revealed distinct metabolic rates for various PL structural components. The glycerol backbone showed the fastest metabolism, while within phospholipid acyl chains, SFAs showed a greater contribution to 13 C labeling compared to MUFAs. Furthermore, we quantified the labeling rates of PL C═C location isomers, highlighting dynamic regulation at the C═C location level. Notably, certain low-abundance n -7 isomers exhibited rapid turnover, suggesting potential functional roles in membrane remodeling or signaling. Analysis of three human breast cancer cell lines (MCF-7, MDA-MB-468, and BT-474) further revealed significant differences in PL metabolism dynamics, with MCF-7 cells showing initially rapid but transient labeling after passage, while MDA-MB-468 cells maintained sustained high fluxes. These findings provide insights into the structural specificity and dynamic regulation of phospholipid metabolism in cancer, offering potential therapeutic implications.