Development of an UPLC–MS/MS method for assaying the enzymatic activity of propionyl coenzyme-A carboxylase

Abstract

Background: Propionyl coenzyme-A carboxylase (PCC) is a mitochondrial enzyme previously quantifiable only by radiometric assay. Herein, we report a UPLC–MS/MS method as a superior alternative method for assaying PCC’s activity. Methodology & Results: For the development of the UPLC–MS/MS method, the mass spectra of propionyl coenzyme-A and methyl malonyl coenzyme-A precursor ions, and their full scan product ions were determined. MRM was used for the quantification of the analytes. The method showed good linearity and selectivity for further bioanalytical study. Conclusion: The developed UPLC–MS/MS method is capable of rapidly quantifying PCC’s enzymatic activity and demonstrated suitability for assaying PCC’s activity in complex biological samples. Thus, the method will be useful in validating recombinant expression of PCC, and potentially for routine quantification of mitochondrial PCC’s activity level in patient cells.