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Simultaneous proteome localization and turnover analysis reveals spatiotemporal coordinations of unfolded protein response

ABSTRACT: The functionality of proteins is dependent on their spatial and temporal distributions, neither of which is directly measured by static protein abundance. Here we report a mass spectrometry-based proteomics workflow and data analysis pipeline, named Simultaneous Proteome Localization and Turnover (SPLAT), to concurrently examine the turnover dynamics and subcellular distributions of whole cell proteomes under perturbation. SPLAT builds on prior work in protein turnover measurements and subcellular localization profiling, by combining dynamic stable isotope labeling, differential ultracentrifugation, and kinetic modeling to concurrently measure changes in protein turnover and subcellular localization under perturbation in one experiment.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Edward Lau

PROVIDER: PXD038054 | JPOST Repository | Sat Dec 31 00:00:00 GMT 2022

REPOSITORIES: jPOST

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Project description: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 post-genome 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-labelling, 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 SILAC, 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 visualised using specialised 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 approximately 20 hours. 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 carboxy 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, multi-protein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.

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Simultaneous proteome localization and turnover analysis reveals spatiotemporal coordinations of unfolded protein response

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