A Quantitative Spatial Proteomics Analysis of Proteome Turnover in Human Cells

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.

Proteomic Analysis of Arginine Methylation Sites in Human Cells Reveals Dynamic Regulation During Transcriptional Arrest

“…Likewise, the overall protein level of hnPRDL was not affected as demonstrated by the unique peptide sequence VFVGGISPDTSEEQIK (Fig. 5C), in full agreement with the previously reported 27 h turnover rate of hnRPDL (64). As a result, our data reveals that the observed down-regulation of MMA on hnRPDL is not because of an increased conversion of MMA into ADMA/SDMA (increased PRMT activity) or altered protein turnover.…”

Cucurbitacin B Alters the Expression of Tumor-Related Genes by Epigenetic Modifications in NSCLC and Inhibits NNK-Induced Lung Tumorigenesis

“…1D, CuB at concentrations of 60 nmol/L or higher significantly increased the mRNA and proteins expression of key cell-cycle regulatory TSGs such as p16 INK4A and p21 CIP1/WAF1 in H1299 cells. However, we found slightly decreased mRNA expressions of these TSGs at 600 nmol/L as compared with 60 nmol/L CuB concentrations, which might be due to the mRNA instability as well as higher rate of protein turnover observed in case of cell-cycle regulators (26,27). Overall, these mRNA and protein expressions are consistently upregulated by the CuB treatment in H1299 cells irrespective of dose responses.…”

Regulation of Fibroblast Growth Factor-inducible 14 (Fn14) Expression Levels via Ligand-independent Lysosomal Degradation

“…2, A and B). In a proteomics study examining protein stability in HeLa cells, it was shown that the average turnover time for HeLa cell proteins is 20 h with more rapid turnover times for proteins involved in dynamic processes such as mitosis, nucleotide binding, and cytoskeletal reorganization (35). Little is known about the steady-state dynamics of the TNFRSF members in the absence of ligand, but such rapid turnover of a cytokine receptor, as exhibited by Fn14 in HeLa cells, seemed quite remarkable.…”