title: Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture.
abstract: Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the "gold standard" for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [(13)C3(15)N1]-pantothenic acid, mammalian cells exclusively incorporated [(13)C3(15)N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [(13)C3(15)N1]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants.
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA; A.J. Drexel Autism Institute, Drexel University, Philadelphia, PA 19104, USA.
authors.forename: Nathaniel W
authors.initials: NW
authors.lastname: Snyder
authors.affiliation: Department of Biology, University of Rochester, Rochester, NY 14627, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Tombline
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Worth
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Parry
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Silvers
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Gillespie
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Sankha S
authors.initials: SS
authors.lastname: Basu
authors.affiliation: Department of Biology, University of Rochester, Rochester, NY 14627, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Millen
authors.affiliation: Department of Biology, University of Rochester, Rochester, NY 14627, USA.
authors.forename: David S
authors.initials: DS
authors.lastname: Goldfarb
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: ianblair@mail.med.upenn.edu.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Blair
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D053898:Biosynthetic Pathways; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D004952:Esters / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D051379:Mice; D010205:Pantothenic Acid / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D004952:Esters; D010205:Pantothenic Acid
keywords: Acetyl-CoA; Coenzyme A; Krebs cycle; Mass spectrometry; Stable isotope labeling; Yeast