Abstract
Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ∼20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.
Highlights
From the ‡Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom; §Division of Biological Chemistry and Drug Discovery, School of Life Sciences Research, College of Life Sciences, University of Dundee; ¶Fingerprints Proteomics facility, College of Life Sciences, University of Dundee
Mass spectrometry-based proteomics has emerged as the technology of choice for studying proteins directly, allowing identification of proteins and post-translational modifications, and quantitative comparisons of how relative protein levels change in cells under different conditions [4]
We have developed a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments
Summary
From the ‡Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom; §Division of Biological Chemistry and Drug Discovery, School of Life Sciences Research, College of Life Sciences, University of Dundee; ¶Fingerprints Proteomics facility, College of Life Sciences, University of Dundee. Protein abundance and the rates of protein synthesis, degradation and turnover have been measured in parallel for whole cells and for separate cytoplasmic, nuclear and nucleolar compartments, providing a cell-based functional annotation of the human proteome.
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