A subcellular map of the human proteome

INTRODUCTION A complete view of human biology can only be achieved by studying the molecular components of its smallest functional unit, the cell. Cells are internally organized into compartments called organelles. The spatial partitioning provided by organelles creates an enclosed environment or surface for chemical reactions tailored to fulfill specific functions. These functions are tightly linked to a specific set of proteins. Therefore, resolving the subcellular location of the human proteome provides information about the function of the organelle and its underlying cellular mechanisms. We present a subcellular map of the human proteome, called the Cell Atlas, to facilitate functional exploration of individual proteins and their role in human biology and disease. RATIONALE Immunofluorescence (IF) microscopy was used to systematically resolve the spatial distribution of human proteins in cultivated cell lines and map them to cellular compartments and substructures with single-cell resolution. This approach allowed definition of the precise location of a majority of the human proteins in their cellular context and exploration of single-cell variations in protein expression patterns. The proteome-wide information about protein spatial distribution was validated with an orthogonal proteomics method, and the results were integrated into existing network models of protein-protein interactions for increased accuracy. RESULTS We report a high-resolution characterization of the spatial subcellular distribution of the human proteome based on more than 80,000 confocal IF images. A total of 12,003 proteins targeted by 13,993 antibodies were classified into one or several of 30 cellular compartments and substructures, altogether defining the proteomes of 13 major organelles. The organelles with the largest proteomes were the nucleus and its substructures (6245 proteins), such as bodies and speckles, and the cytosol (4279 proteins). However, smaller organelles such as the midbody, rods and rings, and nucleoli also showed a larger diversity than previously recognized. Intriguingly, about half of all proteins were localized to multiple compartments, showing that there is a shared pool of proteins even among functionally unrelated organelles. Single-cell analysis revealed 1855 proteins with variation in their expression pattern, either in terms of expression levels or spatial distribution. Last, the spatial information was used to refine biological networks. Our location-pruned network that restricts protein interaction to the same organelle improved the accuracy of the human interactome model. The analysis also included transcriptomics data for all putative protein-coding genes (19,628) in 56 human cell lines of various origins. On average, cell lines expressed 11,490 genes, with half of them (6295) being expressed across all samples, suggesting a “housekeeping” role. CONCLUSION The cellular proteome is compartmentalized and spatiotemporally regulated to a high degree. The high-resolution subcellular map of the human proteome that we provide describes this cellular complexity, with many multilocalizing proteins and single-cell variations. The map is presented as an interactive database called the Cell Atlas, part of the Human Protein Atlas (www.proteinatlas.org). The Cell Atlas constitutes a key resource for a holistic understanding of the human cell and its complex underlying molecular machinery, as well as a major step toward modeling the human cell.