Advances in single-cell metabolomics based on mass spectrometry

Cellular heterogeneity, a crucial factor contributing to the diversity and complexity of biological systems, plays a prominent role in processes such as the tumor microenvironment, immune responses, and stem cell differentiation. The emergence of single-cell omics technologies, encompassing single-cell genomics, proteomics, and metabolomics, has provided powerful tools for exploring cellular heterogeneity. Metabolites, being the downstream products of gene and protein action networks, offer a direct reflection of cellular functional changes in real time. Consequently, metabolomics is regarded as the omics platform most closely aligned with biological phenotypes. Single-cell metabolomics currently enables the analysis of various small molecules, including amino acids, lipids, nucleotides, and their derivatives, to uncover metabolic heterogeneity at the single-cell level. However, the field of single-cell metabolomics faces several technical challenges, including the small size of cells, the complexity of metabolite structure identification, and the low intracellular content of metabolites. Mass spectrometry (MS) has emerged as the primary tool in single-cell metabolomics due to its high sensitivity and resolution, enabling the detection of a wide range of metabolites within individual cells and providing detailed insights into cellular metabolism and function. To address diverse research needs, single-cell metabolomics technologies based on mass spectrometry are categorized into various types. This categorization is based on factors such as spatial resolution, ionization mode, sampling method, and experimental throughput. Among all the developed methods, MS imaging and direct electrospray ionization-based MS analysis are widely employed for single-cell analysis, which will be overviewed in this article.MS imaging provides spatial distribution information of metabolites within tissues or cells. Matrix-assisted laser desorption/ionization MS imaging (MALDI-MSI) is renowned for its simple sample preparation and rapid scanning capabilities, although its spatial resolution remains an area for improvement. New technologies, such as MALDI-2, secondary ion MS, and vacuum ultraviolet desorption ionization MS, have attained higher spatial resolution by employing advanced ionization techniques and innovative light sources. Furthermore, the integration of machine learning and advanced image analysis has enabled accurate identification of cell boundaries and molecular characterization at the single-cell level, significantly advancing the capabilities of MS imaging in single-cell metabolomics.In situ sampling combined with direct electrospray ionization using capillary allows for the direct analysis of metabolites from living cells, offering high ionization efficiency and flexible sampling. This approach has demonstrated considerable success in the study of neuronal function, organelle metabolic profiling, and the analysis of metabolic heterogeneity in complex three-dimensional cell models. However, the sampling process requires skilled operation, and its low throughput presents a challenge. The development of automated sampling systems remains a key area of focus for further improving this technology and increasing its applicability in single-cell metabolomics research.Despite the rapid advancements in these technologies, several challenges persist, including the need for improved annotation of metabolites at the single-cell level, the development of new databases and algorithms to enhance data accuracy, and ensuring the reliability of results. Moreover, integrating single-cell metabolomics with other single-cell omics technologies, such as proteomics and transcriptomics, holds great promise. This multi-omics approach could provide a more comprehensive map of cellular function, revealing intricate interactions between metabolic networks, gene expression, and protein modifications. This integration represents a promising direction for future research in the field of single-cell metabolomics. With ongoing advancements in single-cell metabolomics, the technology is poised to have a profound impact on biology and medicine. It will aid in uncovering key biological processes, accelerating drug development, advancing diagnostic and therapeutic methods, and providing personalized metabolic information for precision medicine, ultimately revolutionizing our understanding of cellular heterogeneity and its implications in health and disease.