Simultaneous quantification of coenzyme A and its salvage pathway intermediates in in vitro and whole cell-sourced samples
We report a method for the simultaneous quantification of the essential metabolic cofactor coenzyme A (CoA) and its thiol-bearing precursors—including 4′-phosphopantetheine, which was recently shown to play a potentially important role as nexus metabolite in CoA biology—with pmol sensitivity. This sensitivity is gained by making use of an established thiol-derivatisation agent that produces a fluorophore upon labeling, which is subsequently separated by HPLC and quantified by fluorescence detection. While previous reports have made use of a similar strategy to quantify CoA, very few have extended the method to the CoA biosynthetic intermediates (some of which occur at levels much lower than CoA) or have critically evaluated its analytical performance. In this study we addressed these shortcomings, and also overcame the difficulty associated with the independent confirmation of the concentrations of the analytical standards used for quantification. The method's utility is showcased through time-course analyses of in vitro reconstituted enzyme reactions and by analysis of extracts from Escherichia coli and Staphylococcus aureus, demonstrating its potential in advancing studies of CoA biosynthesis and CoA-dependent biology in a wide range of systems.