Stable Isotope Labeling with Essential Nutrients in Cell Culture (SILEC): adapting SILAC methodology for small molecule analysis

摘要

Stable isotope standards for coenzyme A (CoASH) and various short chain acyl-CoA molecules were biosynthetically generated with greater than 99percent isotopic purity by passaging murine hepatocytes in media containing [~(13)C_(3),~(15)N]-pantothenate. Charcoal-stripped FBS was more optimal than dialyzed FBS for this application, due to its lower pantothenate content. A stable isotope dilution LC-ESI-MS/MS method using these biosynthetically generated standards was validated and used to measure changes in acyl-CoA species in vitro. Current and future work includes applying this method to other short, medium and long chain acyl-CoA species in cell culture, as well as using the isotopically labeled internal standards to quantitate CoA species extracted from mouse liver and human PBMCs.