Three‐dimensional quantitative mass spectrometry imaging in complex system: From subcellular to whole organism

Abstract Mass spectrometry imaging (MSI) has been applied for label‐free three‐dimensional (3D) imaging from position array across the whole organism, which provides high‐dimensional quantitative data of inorganic or organic compounds that may play an important role in the regulation of cellular signaling, including metals, metabolites, lipids, drugs, peptides, and proteins. While MSI is suitable for investigation of the spatial distribution of molecules, it has a limitation with visualization and quantification of multiple molecules. 3D‐MSI, however, can be applied toward exploring metabolic pathway as well as the interactions of lipid–protein, protein–protein, and metal–protein in complex systems from subcellular to the whole organism through an untargeted methodology. In this review, we highlight the methods and applications of MS‐based 3D imaging to address the complexity of molecular interaction from nano‐ to micrometer lateral resolution, with particular focus on: (a) common and hybrid 3D‐MSI techniques; (b) quantitative MSI methodology, including the methods using a stable isotope labeling internal standard (SILIS) and SILIS‐free approaches with tissue extinction coefficient or virtual calibration; (c) reconstruction of the 3D organ; (d) application of 3D‐MSI for biomarker screening and environmental toxicological research. 3D‐MSI quantitative analysis provides accurate spatial information and quantitative variation of biomolecules, which may be valuable for the exploration of the molecular mechanism of the disease progresses and toxicological assessment of environmental pollutants in the whole organism. Additionally, we also discuss the challenges and perspectives on the future of 3D quantitative MSI.