Techniques for the Measurement of Molecular Species of Acyl-CoA in Plants and Microalgae

The acyl-CoA pool is pivotal in cellular metabolism. The ability to provide reliable estimates of acyl-CoA abundance and distribution between molecular species in plant tissues and microalgae is essential to our understanding of lipid metabolism and acyl exchange. Acyl-CoAs are typically found in low abundance and require specific methods for extraction, separation and detection. Here we describe methods for acyl-CoA extraction and measurement in plant tissues and microalgae, with a focus on liquid chromatography hyphenated to detection techniques including ultraviolet (UV), fluorescence and mass spectrometry (MS). We address the resolution of isobaric species and the selection of columns needed to achieve this, including the analysis of branched chain acyl-CoA thioesters. For MS analyses, we describe diagnostic ions for the identification of acyl-CoA species and how these can be used for both discovery of new species (data dependent acquisition) and routine quantitation (triple quadrupole MS with multiple reaction monitoring).