Subcellular proteomics. Author Josie Christopher, Charlotte Stadler, Claire Martin, Marcel Morgenstern, Yanbo Pan, Cora Betsinger, David Rattray, Diana Mahdessian, Anne-Claude Gingras, Bettina Warscheid, Janne Lehtiö, Ileana Cristea, Leonard Foster, Andrew Emili, Kathryn Lilley Publication Year 2021 Type Journal Article Abstract The eukaryotic cell is compartmentalized into subcellular niches, including membrane-bound and membrane-less organelles. Proteins localize to these niches to fulfil their function, enabling discreet biological processes to occur in synchrony. Dynamic movement of proteins between niches is essential for cellular processes such as signalling, growth, proliferation, motility and programmed cell death, and mutations causing aberrant protein localization are associated with a wide range of diseases. Determining the location of proteins in different cell states and cell types and how proteins relocalize following perturbation is important for understanding their functions, related cellular processes and pathologies associated with their mislocalization. In this Primer, we cover the major spatial proteomics methods for determining the location, distribution and abundance of proteins within subcellular structures. These technologies include fluorescent imaging, protein proximity labelling, organelle purification and cell-wide biochemical fractionation. We describe their workflows, data outputs and applications in exploring different cell biological scenarios, and discuss their main limitations. Finally, we describe emerging technologies and identify areas that require technological innovation to allow better characterization of the spatial proteome. Journal Nat Rev Methods Primers Volume 1 Date Published 2021 ISSN Number 2662-8449 DOI 10.1038/s43586-021-00029-y Alternate Journal Nat Rev Methods Primers PMCID PMC8451152 PMID 34549195 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML