Mass-spectrometry-based methods for relative proteome quantification have broadly affected life science research. However, important research directions, particularly those involving mathematical modelling and simulation of biological processes, also critically depend on absolutely quantitative data—that is, knowledge of the concentration of the expressed proteins as a function of cellular state. Until now, absolute protein concentration measurements of a considerable fraction of the proteome (73%) have only been derived from genetically altered Saccharomyces cerevisiae cells1, a technique that is not directly portable from yeast to other species. Here we present a mass-spectrometry-based strategy to determine the absolute quantity, that is, the average number of protein copies per cell in a cell population, for a large fraction of the proteome in genetically unperturbed cells. Applying the technology to the human pathogen Leptospira interrogans, a spirochete responsible for leptospirosis2, we generated an absolute protein abundance scale for 83% of the mass-spectrometry-detectable proteome, from cells at different states. Taking advantage of the unique cellular dimensions of L. interrogans, we used cryo-electron tomography morphological measurements to verify, at the single-cell level, the average absolute abundance values of selected proteins determined by mass spectrometry on a population of cells. Because the strategy is relatively fast and applicable to any cell type, we expect that it will become a cornerstone of quantitative biology and systems biology.
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This project has been funded in part by ETH Zurich, the Swiss National Science Foundation (grant 31000-10767), federal funds from the National Heart, Lung and Blood Institute, the National Institutes of Health (contract no. N01-HV-28179), SystemsX.ch, the Swiss initiative for systems biology, in part by the PROSPECTS (proteomics in time and space) European network of excellence, and with funds from the ERC project ‘Proteomics V3.0’ for R.A. J.M. was supported by a fellowship from the Swedish Society for Medical Research (SSMF), M.B. was supported by a long-term fellowship of the European Molecular Biology Organization and a Marie Curie fellowship of the European Commission, A.S. and V.L. were supported by the Competence Center for Systems Physiology and Metabolic Diseases. We thank O. Medalia and the electron microscopy facility of ETH Zurich (EMEZ) for support, and D. A. Haake for critical reading of the manuscript.
Author Contributions J.M. and M.B. planned the experiments, performed the experimental work and data analysis and wrote the manuscript. A.S. and V.L. participated in the experimental work and the data analysis and E.W.D. assembled the PeptideAtlas build. R.A. was the project leader and wrote the manuscript.
The mass spectrometry data, including spectra and the identified peptides and proteins, have been deposited into a PeptideAtlas instance found at http://www.peptideatlas.org/builds/ (Leptospira interrogans, Jan 2008 Build).
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Malmström, J., Beck, M., Schmidt, A. et al. Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans. Nature 460, 762–765 (2009). https://doi.org/10.1038/nature08184
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