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Mol Cell Proteomics. 2016 Dec;15(12):3551-3563. Epub 2016 Oct 20.

Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing.

Author information

1
From the ‡University of Rochester Mass Spectrometry Resource Laboratory, Rochester, NY.
2
§Department of Biology, University of Rochester, Rochester, NY.
3
¶Department of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY.
4
From the ‡University of Rochester Mass Spectrometry Resource Laboratory, Rochester, NY; sghaemma@bio.rochester.edu.

Abstract

Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. The results indicate that upon reaching quiescence, fibroblasts compensate for lack of cellular growth by globally downregulating protein synthesis and upregulating protein degradation. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data.

PMID:
27765818
PMCID:
PMC5141271
DOI:
10.1074/mcp.M116.063230
[Indexed for MEDLINE]
Free PMC Article

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