Minimally Invasive Single-Cell Metabolomics Analysis to Unveil Different Trajectories of Individual Cells in Ferroptosis

Existing cellular heterogeneity necessitates continuous analysis in a single cell to capture dynamic information. However, current mass spectrometry-based single-cell metabolomics analysis is a destructive technique and cannot perform multiple samplings from the same cell, which complicates the accurate tracking of metabolite level alterations. In this study, we present a minimally invasive single-cell mass spectrometry technique that enables sampling of less than 10% of the total cell volume per iteration by using nanocapillaries. This minimally invasive process allows for repeated sampling from a single cell without compromising its essential physiological functions. The resulting time-resolved metabolomics analysis provides the alteration in metabolite levels in a single cell in a process of erastin-induced ferroptosis. As compared to the metabolite levels at certain time points, the alteration in metabolite levels exhibits a stronger correlation with the extent of mitochondrial lipid peroxidation. More importantly, by mapping the differentiation trajectory of individual cells during this process, two distinct groups─proliferating and quiescent cells─are identified. These two groups of cells exhibit different sensitivities to ferroptosis, with quiescent cells being more susceptible to ferroptosis, which could only be tracked by using the time-resolved single-cell metabolomics analysis. Ultimately, this technique overcomes the challenge in single-cell dynamic studies posed by cellular heterogeneity, providing more accurate biological insights into the diversity and complexity of cellular metabolic activities.