Deep Profiling of Aminophospholipids Reveals a Dysregulated Desaturation Pattern in Breast Cancer Cell Lines

Phosphatidylethanolamines (PEs), ether-PEs, and phosphatidylserines (PSs) are glycerophospholipids harboring a primary amino group in their headgroups. They are key components of mammalian cell membranes and play pivotal roles in cell signaling and apoptosis. In this study, a liquid chromatography–mass spectrometry (LC–MS) workflow for deep profiling of PEs, ether-PEs, and PSs has been developed by integrating two orthogonal derivatizations: (1) derivatization of the primary amino group by 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) for enhanced LC separation and MS detection and (2) the Paternò–Büchi (PB) reaction for carbon–carbon double bond (C═C) derivatization and localization. Significant improvement of the limit of identification down to the C═C location has been achieved for the standards of PSs (3 nM) and ether-PEs (20 nM). This workflow facilitates an identification of more than 200 molecular species of aminophospholipids in the porcine brain, two times more than those identified without TMAB-NHS derivatization. Importantly, we discovered that the n-10 isomers in C16:1 and C18:1 of aminophospholipids showed elevated contribution among other isomers, which correlated well with an increased transcription of the corresponding desaturase (FADS2) in the human breast cancer cell line (MDA-MB-231) relative to that in the normal cell line (HMEC). The abovementioned data suggest that lipid reprograming via forming different C═C location isomers might be an alternative mechanism in cancer cells.

• This study presents a liquid chromatography-mass spectrometry (LC-MS) workflow for sensitive and in-depth profiling of aminophospholipids by integrating two orthogonal derivatization strategies: amine derivatization with 4-trimethylammoniumbutyryl-N-hydroxysuccinimide (TMAB-NHS) to enhance LC separation and MS detection, and C=C derivatization via the Paternò-Büchi (PB) reaction for C=C location analysis.
• It is found that elevated n-10 C=C location isomers in C16:1 and C18:1 of aminophospholipids in the human breast cancer cell line are correlated with increased transcription of FADS2 desaturase, uncovering lipid reprogramming based on alternative desaturation in cancer cells.