title: Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing.
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.
authors.affiliation: From the ‡University of Rochester Mass Spectrometry Resource Laboratory, Rochester, NY.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Welle
authors.affiliation: §Department of Biology, University of Rochester, Rochester, NY.
authors.forename: Tian
authors.initials: T
authors.lastname: Zhang
authors.affiliation: From the ‡University of Rochester Mass Spectrometry Resource Laboratory, Rochester, NY.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Hryhorenko
authors.affiliation: ¶Department of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY.
authors.forename: Shichen
authors.initials: S
authors.lastname: Shen
authors.affiliation: ¶Department of Pharmaceutical Sciences, University at Buffalo, Buffalo, NY.
authors.forename: Jun
authors.initials: J
authors.lastname: Qu
authors.affiliation: From the ‡University of Rochester Mass Spectrometry Resource Laboratory, Rochester, NY; sghaemma@bio.rochester.edu.|§Department of Biology, University of Rochester, Rochester, NY.
authors.forename: Sina
authors.initials: S
authors.lastname: Ghaemmaghami
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome