Single-cell structural lipidomics using a miniature dual-LIT mass spectrometer

Structural lipidomics provides comprehensive information on lipidomes, and there is great interest in applying it to single-cell analysis for accurate cell phenotyping and lipid pathway studies. However, structural lipidomics relies on tandem mass spectrometry analysis of a large number of lipid species, which remains challenging for single-cell analysis due to the small sample amounts. Herein, using a miniature dual-linear ion trap (LIT) mass spectrometer with enhanced ion processing capability, we developed an effective strategy to achieve annotations of more than 100 lipid species in a single cell with high structural specificity. The ion utilization efficiency was greatly improved with multi-stage MS ^<i>n</i> (<i>n</i> = 2-4) analysis performed for each lipid species to acquire structural information at different levels. From a single MDA-MB-468 cancer cell, we identified 100+ lipids, including 64 lipids at the acyl-chain sum composition level, 23 at the <i>sn</i>-position level, 30 at the C[double bond, length as m-dash]C location level, and 20 at the C[double bond, length as m-dash]C/<i>sn</i>-position level, primarily phosphatidylcholamines (PCs) and phosphatidylethanolamines (PEs). Significant variations in <i>sn</i>-position and C[double bond, length as m-dash]C location isomers were observed in doxorubicin-resistant and -sensitive K562 cells. With enriched information at the structural lipidomics level, the correlation was established between variations in lipidome and response to ferroptosis for human cancer cells.