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Turnover rates of human muscle proteins in vivo reported in fractional, mole and absolute units

ABSTRACT: Protein fractional turnover rates (FTR) represent measurements of flux through a protein pool, i.e. net abundance (ABD) of the protein. If protein abundance is not measured or is different between experimental conditions the interpretation of FTR data may be confounded. This project investi-gates the consequences of reporting turnover rates of human muscle proteins in vivo in mole and absolute units (that incorporate protein abundance data) compared to fractional (%/d) data that ignore protein abundance. Three physically active males (21 ± 1 years) were recruited and un-derwent a 12-d protocol of daily deuterium oxide (D2O) consumption and biopsies of vastus lat-eralis on days 8 and 12. Protein abundances were normalised to yeast alcohol dehydrogenase, added during sample preparation, and FTR was calculated from time-dependent changes in pep-tide mass isotopomer profiles. FTR and abundance data (fmol/ μg protein) were combined to calculate molar turnover rates (MTR; fmol/ μg protein/ d) and absolute turnover rates (ATR; ng/ d). Abundance data were collected for 1,772 proteins and FTR data were calculated from 3,944 peptides representing 935 proteins (average 3 peptides per protein). The median (M), lower- (Q1) and upper-quartile (Q3) values for protein FTR (%/d) were M = 4.39, Q1 = 0.96, Q3 = 7.84. Our analyses suggest MTR data is preferred over FTR, particularly for studies on multiprotein com-plexes, wherein MTR takes account of potential differences amongst the molecular weight of the component subunits. ATR data may be preferred over MTR and FTR, particularly when compar-ing samples with different abundance profiles.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Muscle, Muscle Cell

SUBMITTER: Jatin Burniston

LAB HEAD: Jatin Burniston

PROVIDER: PXD046509 | Pride | 2025-06-05

REPOSITORIES: Pride

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			Action	DRS
	Pepdite_identifications_myofibrillar_fraction.xlsx	Xlsx
	Pepdite_identifications_soluble_fraction.xlsx	Xlsx
	X_D08.raw.raw	Raw
	X_D08_MS_Only-M.raw.zip	Raw
	X_D12.raw.raw	Raw

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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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Turnover rates of human muscle proteins in vivo reported in fractional, mole and absolute units

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