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

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.