Development of a Chemoplexed LC-MS Method for Quantification of the CYP 1A2 Enzyme System, presented at CPSA 2013, Oct. 7-10, 2013 in Langhorne, PA.

Cytochrome P450 (CYP) family of enzymes are the primary enzymes responsible for metabolism of xenobiotics. Given the importance of these enzymes it is clear why CYP catalytic efficiency is important in pre-clinical drug development. Unfortunately, consistently measuring the catalytic efficiency of enzyme-containing reagents such as liver microsomes has proved challenging. Activity assays are subject to the experimental conditions and direct protein measurements are not typically done.
We have developed a quantitative method for the determination of substrate, metabolite and enzyme in the same assay which allows us to make measurements of the complete enzyme system in a single analytical event. CYP1A2 activity was determined by metabolism of Phenacetin to Acetaminophen, and CYP1A2 protein concentration was measured via a surrogate peptide approach in a combined assay using LC-MS/MS detection. Results from a small data set show a good correlation between enzyme concentration and activity.
Our approach shows promise as a mass spectrometry-based analytical tool for quantifying complete enzyme systems rather than individual components. The ability to multiplex assays involving separate chemotypes such as small molecule substrates, metabolites and cofactors along with proteins and peptides in the same samples using a common analytical method may prove to be a valuable tool for studies in systems biology. Practical mass spectrometry-based, chemo-plexed analyses may open the door to the use of enzyme systems as biomarkers and clinical diagnostic tools.


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