A Liquid Chromatography-Mass Spectrometry Method to Quantify Total Coenzyme A Concentration and Isotopic Labeling

Abstract Coenzyme A is an essential cofactor synthesized from pantothenate, which carries activated acyl groups in cellular metabolic reactions. Coenzyme A participates in catabolism of carbohydrates, fats, and amino acids; biosynthesis of fatty acids, cholesterol, and heme; and protein modification, including acetylation and 4-phosphopantetheinylation. Despite CoA’s critical functions, the regulation of CoA levels and the rate of CoA synthesis in different cell types and disease states are not well understood. Acyl-CoA species are challenging to measure due to instability, poor ionization, and the wide range of biochemical properties conferred by different acyl chain lengths; moreover, most current methods cannot measure CoA isotopic labeling, required to quantify the CoA synthesis rate or to measure concentration using isotope-labeled internal standards. Here, we describe a method to quantify the concentration and isotopic labeling of total CoA, defined as the sum of CoASH plus all acyl-CoA species. Acyl-CoA species are hydrolyzed using sodium hydroxide to remove acyl chains; then, CoA is derivatized with N-ethylmaleimide (NEM). Following protein precipitation and solid-phase extraction, samples are analyzed by liquid chromatography-mass spectrometry. This method is linear in a wide range that captures mouse tissue CoA levels, with accuracy within 15% error and precision below 15% relative standard deviation for standards and tissue samples. We applied this method to measure total CoA concentration in five mouse tissues and the total CoA synthesis rate in mouse liver via infusion of 13C–15N-pantothenate. Overall, this method offers a tractable approach to measure total CoA concentration and synthesis rate in health and disease.