Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.

Here we present a spatiotemporal dissection of proteome single cell heterogeneity in human cells, performed with subcellular resolution over the course of a cell cycle. We identify 17% of the human proteome to display cell-to-cell variability, of which we could attribute 25% as correlated to cell cycle progression, and present the first evidence of cell cycle association for 258 proteins. A key finding is that the variance, of many of the cell cycle associated proteins, is only partially explained by the cell cycle, which hints at cross-talk between the cell cycle and other signaling pathways. We also demonstrate that several of the identified cell cycle regulated proteins may be clinically significant in proliferative disorders. This spatially resolved proteome map of the cell cycle, integrated into the Human Protein Atlas, serves as a valuable resource to accelerate the molecular knowledge of the cell cycle and opens up novel avenues for the understanding of cell proliferation.

Mitochondria is involved in a numerous variety of cellular functions beyond its role in energy metabolism. Defining the human mitochondrial proteome is crucial to understand the mitochondria’s diverse functions and role in disease. Here, we present an image-based map of the human mitochondrial proteome containing 1,098 proteins. The single cell resolution revealed extensive heterogeneity for as much as 20% (n=226) of the mitochondrial proteome.  These variations are independent of cell cycle position and likely represent metabolic fluctuations in the cell. Our analysis shows that 48% (n=524) of the proteins localize to additional cellular compartments, further contributing to the diverse cellular functions of mitochondria. This map of the mitochondrial proteome, part of the Cell Atlas of the Human Protein Atlas database (www.proteinatlas.org), provides a valuable knowledge resource for studies of mitochondria function, dysfunction and disease.

In the interphase cell, the membrane-less nucleoli are the sites of ribosome biogenesis. As part of the Human Protein Atlas we created an image catalogue comprising 1,314 nucleolar proteins using antibody-based proteomics. We show experimental evidence for 1,027 proteins localizing to the whole nucleoli and 287 to the fibrillar center or dense fibrillar component. We also propose a new sub-compartment located in the nucleoplasmic border denoted as nucleoli rim, comprising at least 131 proteins. As a step toward better understanding of nucleolar protein function during cell division, we additionally generated confocal images of 68 nucleolar proteins being recruited to the chromosomal periphery in mitosis. Thanks to the single cell resolution we were able to define three expression phenotypes among the mitotic chromosome proteins; early, intermediate and late recruitment suggesting phase specific functions. We also for the first time provide a proteome-wide confirmation that the nucleoli in general, but mitotic chromosome proteins in particular have a higher predicted intrinsic disorder level compared to cytoplasmic proteins, indicating that the perichromosomal layer indeed is a liquid-like layer.