Updated publication reference for DOI(s): 10.1038/s41586-020-2402-x. Updated publication reference for PubMed record(s): 32555458. Updated publication reference for DOI(s): 10.1038/s41586-020-2402-x. Proteins preform the vast majority of functions in all biological domains but their large-scale investigation has lagged behind for technological reasons. Since the first essentially complete eukaryotic proteome was reported1, advances in mass spectrometry (MS)-based proteomics2 have enabled increasingly comprehensive identification and quantification of the human proteome3456. However, there are few comparisons across species, especially compared to genomics initiatives7. Here, we employ an advanced proteomics workflow, in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system, for the in-depth measurement of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to the scientific community. The data also provide a foundation for machine learning, as we demonstrate by experimentally confirming predicted peptide properties of bacteroides uniformis. Our results provide a comparative view into the functional organization of organisms across the entire evolutionary range. A remarkably high fraction of the total proteome mass in all kingdoms is dedicated to protein homeostasis and folding, highlighting the challenge of maintaining protein structure across all of life. Likewise, a constantly high fraction is involved in supplying energy resources, although the pathways range from photosynthesis through iron sulphur metabolism to carbohydrate metabolism.