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Proteomics. 2016 Aug;16(15-16):2106-17. doi: 10.1002/pmic.201500433. Epub 2016 Jul 8.

Large-scale multiplex absolute protein quantification of drug-metabolizing enzymes and transporters in human intestine, liver, and kidney microsomes by SWATH-MS: Comparison with MRM/SRM and HR-MRM/PRM.

Author information

1
Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
2
Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
3
AMED-CREST, Japan Agency for Medical Research and Development, Tokyo, Japan.
4
Department of Drug Metabolism and Pharmacokinetics, Drug Safety Research Center, Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd, Tokushima, Japan.
5
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

Abstract

The purpose of the present study was to examine simultaneously the absolute protein amounts of 152 membrane and membrane-associated proteins, including 30 metabolizing enzymes and 107 transporters, in pooled microsomal fractions of human liver, kidney, and intestine by means of SWATH-MS with stable isotope-labeled internal standard peptides, and to compare the results with those obtained by MRM/SRM and high resolution (HR)-MRM/PRM. The protein expression levels of 27 metabolizing enzymes, 54 transporters, and six other membrane proteins were quantitated by SWATH-MS; other targets were below the lower limits of quantitation. Most of the values determined by SWATH-MS differed by less than 50% from those obtained by MRM/SRM or HR-MRM/PRM. Various metabolizing enzymes were expressed in liver microsomes more abundantly than in other microsomes. Ten, 13, and eight transporters listed as important for drugs by International Transporter Consortium were quantified in liver, kidney, and intestinal microsomes, respectively. Our results indicate that SWATH-MS enables large-scale multiplex absolute protein quantification while retaining similar quantitative capability to MRM/SRM or HR-MRM/PRM. SWATH-MS is expected to be useful methodology in the context of drug development for elucidating the molecular mechanisms of drug absorption, metabolism, and excretion in the human body based on protein profile information.

KEYWORDS:

Biomedicine; CYP; Microsomes; SWATH-MS; Transporter; UGT

PMID:
27197958
DOI:
10.1002/pmic.201500433
[Indexed for MEDLINE]

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