Automated single-cell analysis by single-cell side-orifice flow cytometry chain electrospray ionization mass...

Single-cell mass spectrometry (SCMS) provides a powerful tool for probing cellular heterogeneity and uncovering biological mechanisms that are often obscured at the population level. However, SCMS faces challenges such as short signal duration and limited tandem mass spectrometry (MS/MS) data for metabolite identification, primarily due to the extremely low sample volume. Previously, we developed the chain electrospray ionization for ultra-low-volume sample analysis. In this paper, a novel single-cell side-orifice flow cytometry combined with the chain electrospray ionization mass spectrometry platform was proposed. This platform enables automated single-cell analysis, successfully identifying 486 lipids from five cell lines (A549, HEK-293T, HCCLM3, MHCC97-H, and SK-HEP-1). Principal component analysis-linear discriminant analysis (PCA-LDA) was employed to accurately differentiate these cell types based on the identified lipid profiles. The platform demonstrates direct, rapid, and high-coverage analysis of single-cell biomolecules, even at extremely low volumes. The novel SCMS not only enhances our understanding of cellular heterogeneity but also holds the potential for identifying disease biomarkers and exploring metabolic pathways in single cells.