Abstract
Quantitative proteomics is an emerging technique with promising applications in the field of drug metabolism to improve the translation of in vitro, preclinical, and individual data to population pharmacokinetics. For example, quantitative proteomics data can be utilized for in vitro-in vivo extrapolation (IVIVE), in vitro model validation, and determination of interindividual and species differences. Here, we describe a detailed methodology for the quantification of cytochrome P450 (CYP) in human samples. The methodology includes the preparation of human liver microsomal samples, selection of specific surrogate peptides, protein digestion, liquid chromatography (LC) separation, and mass spectrometry (MS) quantification. The focus of this chapter is limited to targeted protein quantification using a conventional LC and a triple quadruple MS instrument in selected reaction monitoring (SRM) mode. In targeted proteomics, the membrane-anchored CYP proteins are digested in-solution to peptides, and the peptide mixtures are separated by reversed-phase LC, and peptide signals are detected by a SRM-based technique (Zhang, Liu, et al. 2011). Peptide standards (light or stable isotope-labeled) are used as calibrator and internal standards, which allows absolute quantification of peptide levels. Stepwise details are provided for the efficient implementation of the quantitative proteomics technique by experts and new researchers.
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Takahashi, R.H., Achour, B., Prasad, B. (2021). Quantitative Determination of Cytochrome P450 Using LC-MS/MS. In: Yan, Z., Caldwell, G.W. (eds) Cytochrome P450. Methods in Pharmacology and Toxicology. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1542-3_3
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DOI: https://doi.org/10.1007/978-1-0716-1542-3_3
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