From workflow optimisation to biological significance: Single-cell LC-MS lipidomics of Caco-2 and HT29-MTX

Caco-2 and HT29-MTX cells are widely used as gut models, showing enterocyte and goblet cell like characteristics. Lipidmics is an informative way to explore the cell composition and metabolic regulation, but bulk analyses diminish cell heterogeneity. In this study, we optimised an LC-MS workflow to profile Caco-2 and HT29-MTX cells at single-cell resolution. Initial experiments using an IPA-based mobile phase showed extensive contamination, with solvent blanks containing hundreds of lipid features, dominated by neutral lipids. To overcome this, an improved workflow employs a phenyl-hexyl LC column with acetonitrile-based mobile phase and a shorter gradient, coupled to Xevo MRT. This reduced background signals, improved sensitivity, and provided more reliable MS/MS-based lipid identifications.