Quantification of CYP2E1 in rat liver by UPLC-MS/MS-based targeted proteomics assay: a novel approach for enzyme activity assessment.

CYP2E1 is one of the most crucial isozymes of CYP450. It is responsible for metabolizing and activating a large number of toxicants and carcinogens, but the correlation between its abundance and activity has not been widely studied. With the flourishing of modern mass spectrometry technology, quantifying complex biological proteins and studying the relationship between their abundance and activity have become practicable. In our study, an accurate, sensitive, and stable LC-MS/MS-based method was developed and validated. The method can accurately quantify the abundance of CYP2E1 in the rat liver microsome and S9 fraction. The quantitative linearity of the method is between 2 and 320 ng/mL, and the run time is 16.5 minutes. Meanwhile, we used the probe substrate method (with chlorzoxazone as the substrate) as a reference to analyze the correlation between its activity and abundance. The result illustrated that the abundance of CYP2E1 by LC-MS/MS has a strong positive correlation with its activity. This is a relationship worth studying, which has not been reported before. We also explored the correlation between quantitative results by traditional methods (western blot and RT-PCR) and activity, and the positive correlation was not obvious. Therefore, when testing the correlation between metabolic enzyme abundance and activity, the LC-MS/MS-based method is confirmed to be more accurate than conventional methods. It will provide a meaningful way of researching the metabolic enzymes in drug interactions. Furthermore, we found that the S9 fraction can also be used for mass spectrometry quantitative analysis, which is helpful for promoting the practical application of targeted protein technology.