title: SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards.
abstract: Stable isotope labeling by essential nutrients in cell culture (SILEC) was recently developed to generate isotopically labeled coenzyme A (CoA) and short-chain acyl-CoA thioesters. This was accomplished by modifying the widely used technique of stable isotope labeling by amino acids in cell culture to include [(13)C(3)(15)N]-pantothenate (vitamin B(5)), a CoA precursor, instead of the isotopically labeled amino acids. The lack of a de novo pantothenate synthesis pathway allowed for efficient and near-complete labeling of the measured CoA species. This protocol provides a step-by-step approach for generating stable isotope-labeled short-chain acyl-CoA internal standards in mammalian and insect cells as well as instructions on how to use them in stable isotope dilution mass spectrometric-based analyses. Troubleshooting guidelines, as well as a list of unlabeled and labeled CoA species, are also included. This protocol represents a prototype for generating stable isotope internal standards from labeled essential nutrients such as pantothenate. The generation and use of SILEC standards takes approximately 2-3 weeks.
authors.affiliation: Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA.
authors.forename: Sankha S
authors.initials: SS
authors.lastname: Basu
authors.forename: Ian A
authors.initials: IA
authors.lastname: Blair
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D003065:Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D004330:Drosophila; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards*; D012015:Reference Standards
chemical_list: D003065:Coenzyme A