Analytical challenges in mapping the subcellular metabolome and lipidome

Mass spectrometry-based metabolomics and lipidomics are central analytical tools for characterizing cellular chemical composition. However, most workflows still rely on the simplifying assumption of homogeneous intracellular pools, which is increasingly inadequate for spatially organized eukaryotic systems. Metabolites and lipids are distributed across subcellular compartments that differ in chemical environment, turnover, and accessibility, thereby affecting both measurement and interpretation. Recent advances in subcellular and spatial metabolomics have highlighted both the potential and the limitations of organelle-resolved analysis, particularly in terms of extraction chemistry, quantification, and data interpretation. In this review, we critically examine organelle-resolved metabolomics and lipidomics from a mass spectrometry-centric perspective, treating subcellular compartmentalization as an analytical variable rather than solely a biological feature. By comparing metabolomics and lipidomics studies on subcellular compartments, we evaluate fractionation-based, affinity-based, and spatial MS strategies, and we highlight current capabilities, common artefacts, and future opportunities, including the integration of stable isotope tracing and emerging single-organelle approaches such as Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) and Direct Organelle Mass Spectrometry (DOMS).