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


CONCLUSIONS
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.
Department of Pharmacy, National University of Singapore, Singapore.
Mahnaz Darvish
MD
Damavandi
Eric Chun Yong
EC
Chan
Jan P
JP
Kraus
Paul Chi-Lui
PC
Ho
Tse Siang
TS
Kang
D000214:Acyl Coenzyme A / Q000378:metabolism; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D016014:Linear Models; D043586:Methylmalonyl-CoA Decarboxylase / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000214:Acyl Coenzyme A; C015357:methylmalonyl-coenzyme A; D043586:Methylmalonyl-CoA Decarboxylase