title: Kinetics of precursor labeling in stable isotope labeling in cell cultures (SILAC) experiments.
abstract: Recent advances in mass spectrometry have enabled proteome-wide analyses of cellular protein turnover. These studies have been greatly propelled by the development of stable isotope labeling in cell cultures (SILAC), a set of standardized protocols, reagents aimed at quantifying the incorporation of (15)N/(13)C labeled amino acids into proteins. In dynamic SILAC experiments, the degree of isotope incorporation in proteins is measured over time and used to determine turnover kinetics. However, the kinetics of isotope incorporation in proteins can potentially be influenced not only by their intracellular turnover but also by amino acid uptake, recycling and aminoacyl-tRNA synthesis. To assess the influence of these processes in dynamic SILAC experiments, we have measured the kinetics of isotopic enrichment within intracellular free amino acid and aminoacyl-tRNA precursor pools in dividing and division-arrested neuroblastoma cells following the introduction of extracellular (15)N labeled amino acids. We show that the total flux of extracellular amino acids into cells greatly exceeds that of intracellular amino acid recycling and synthesis. Furthermore, in comparison to internal sources, external amino acids are preferentially utilized as substrates for aminoacyl-tRNA precursors for protein synthesis. As a result, in dynamic SILAC experiments conducted in culture, the aminoacyl-tRNA precursor pool is near completely labeled in a few hours and protein turnover is the limiting factor in establishing the labeling kinetics of most proteins.
authors.affiliation: Department of Biology, University of Rochester , Rochester, New York 14627, United States.
authors.forename: Tian
authors.initials: T
authors.lastname: Zhang
authors.forename: John C
authors.initials: JC
authors.lastname: Price
authors.forename: Eslam
authors.initials: E
authors.lastname: Nouri-Nigjeh
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Marc K
authors.initials: MK
authors.lastname: Hellerstein
authors.forename: Jun
authors.initials: J
authors.lastname: Qu
authors.forename: Sina
authors.initials: S
authors.lastname: Ghaemmaghami
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D018929:Cell Culture Techniques; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007553:Isotope Labeling; D007700:Kinetics; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
chemical_list: D000596:Amino Acids; D012346:RNA, Transfer, Amino Acyl