Simultaneous In-Depth Single-Cell Proteomic and Metabolomic Analysis

Simultaneous characterization of proteomic and metabolomic profiles at the single-cell level is crucial for deciphering cellular heterogeneity and elucidating disease mechanisms. However, it is still a great challenge to achieve high-depth dual-omics analysis in the same single cell. Here, we propose a unified strategy called one-shot hybrid-mode single-cell proteome and metabolome analysis (hybrid-scPMA), in which the mass spectrometry (MS) detection mode of data-independent acquisition (DIA) is utilized for the analysis of peptides from protein digestion, and the data-dependent acquisition (DDA) mode is used for metabolite analysis in a single liquid chromatography-mass spectrometry (LC-MS) analysis run, enabling deep analysis of the proteome and metabolome in single-cell samples. Building upon the strategy, we established an improved single-cell multiomics analysis workflow that integrated automated single-cell capture, simplified sample pretreatment, LC injection and separation, and DIA-DDA hybrid-mode MS detection. With this approach, we achieved an average identification of 3510 protein groups and 255 metabolites from single HepG2 cells, representing a substantial increase in identification depth over previous approaches. We also performed time-resolved proteomic and metabolomic profiling of HepG2 single cells undergoing sorafenib drug intervention, resolving drug response characteristics at the single-cell level and providing multiomics insights into drug mechanisms from a proteo-metabolomic perspective.