Abstract
Most P450 protein quantitation methods involved the time-consuming preparation of microsomes and therefore are not amenable for high-throughput analysis. We here report a new method to measure P450 CYP3A4 protein levels directly from cell lysates. A direct sample preparation method from hepatocyte cell lysate has been developed for the quantification of CYP3A4 protein levels by combining a modified semi-automated precipitation with a filter-aided sample preparation. This novel LC-MS/MS-based method provides simple, subfemtomole sensitivity and rapid quantitation of CYP3A4 protein levels directly from hepatocyte lysate without the need for microsome preparation. A rapid, accurate and sensitive method has been developed and implemented to quantify CYP3A4 protein in hepatocytes down to 0.05 million cells in CYP induction studies. The number of cells required for quantitation was well below the typical 0.25 million cells used in a CYP induction study.
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