Opportunities and challenges for sample preparation and enrichment in mass spectrometry for single‐cell metabolomics

Abstract Single‐cell heterogeneity in metabolism, drug resistance and disease type poses the need for analytical techniques for single‐cell analysis. As the metabolome provides the closest view of the status quo in the cell, studying the metabolome at single‐cell resolution may unravel said heterogeneity. A challenge in single‐cell metabolome analysis is that metabolites cannot be amplified, so one needs to deal with picolitre volumes and a wide range of analyte concentrations. Due to high sensitivity and resolution, MS is preferred in single‐cell metabolomics. Large numbers of cells need to be analysed for proper statistics; this requires high‐throughput analysis, and hence automation of the analytical workflow. Significant advances in (micro)sampling methods, CE and ion mobility spectrometry have been made, some of which have been applied in high‐throughput analyses. Microfluidics has enabled an automation of cell picking and metabolite extraction; image recognition has enabled automated cell identification. Many techniques have been used for data analysis, varying from conventional techniques to novel combinations of advanced chemometric approaches. Steps have been set in making data more findable, accessible, interoperable and reusable, but significant opportunities for improvement remain. Herein, advances in single‐cell analysis workflows and data analysis are discussed, and recommendations are made based on the experimental goal.