Single-Cell Lipidomics: Performance Evaluation Across Four Liquid Chromatography Mass Spectrometry (LC-MS) Systems

Single-cell lipidomics holds tremendous promise for understanding a wide range of pathological conditions involving heterogenous cell populations, including infection, cancer, diabetes, and cardiovascular disease and yet its widespread adoption has been hitherto limited. Although Liquid Chromatography Mass Spectrometry (LC-MS) is a globally established method for lipidomics, its application to single cells has been considered particularly challenging, if not impossible, due to the very low sample volume and the high dynamic range and structural complexity of cellular lipids. Recent advances have shown that LC-MS-based single-cell lipidomics is achievable, offering the benefit of sampling cells in their native state, as well as chromatographic separation to reduce matrix effects and enhance peak annotation. In this study, we advocate for wider adoption of single-cell lipidomics by demonstrating that a range of widely accessible LC-MS platforms can successfully generate single-cell lipid profiles. Using four distinct instrumental configurations, we provide a perspective on the achievable depth of coverage and annotation. We show that polarity switching, ion mobility spectrometry, and electron-activated dissociation significantly enhance both lipidome coverage and confidence in lipid identification from single cells.