Quantification of Sulfotransferases 1A1 and 1A3/4 in Tissue Fractions and Cell Lines by Multiple Reaction Monitoring Mass Spectrometry.

Drug metabolism letters Pub Date : 2017-11-17 DOI:10.2174/1872312811666170731170153

Sho Yoshitake, Melissa McKay-Daily, Masaki Tanaka, Zeqi Huang

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引用次数: 2

Abstract

Background: Within the sulfotransferase (SULT) superfamily of metabolic enzymes, SULT1A1 and 1A3/4 isoforms are of particular interest, due to their abilities to catalyze the sulfation of phenolic endobiotics and xenobiotics. Although the difference in their substrate specificity is well documented, an isoform-specific quantification method is still not available.

Objective: To detect and quantify SULT1A1 and 1A3/4 in S9 fractions and cell lines using targeted mass spectrometry-based proteomics.

Method: Samples were tryptically digested, and signature peptides were quantified using liquid chromatography- multiple reaction monitoring mass spectrometry (LC-MRM/MS). Stable isotopelabeled (SIL) peptides were used as internal and calibration standards. SULT1A1 and SULT1A3/4 were quantified in various S9 fractions and cell line samples.

Results: Intraday and interday variabilities were low for relative quantification in S9 and cell line matrices (<8%). Expression profiles were validated using Western blot analysis of S9 fractions and lentiviral transduced SULT1A-overexpressing cell lines.

Conclusion: A reproducible method for simultaneous quantification of SULT1A1 and SULT1A3/4 in S9 fractions and cell line samples was established and validated.

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多反应监测质谱法测定组织和细胞系中硫转移酶1A1和1A3/4的含量。

背景:在代谢酶的硫转移酶(SULT)超家族中，SULT1A1和1A3/4异构体由于其催化酚类内源性和外源性磺化的能力而受到特别关注。虽然它们的底物特异性的差异是有目共知的，但一种异构体特异性的定量方法仍然不可用。目的:利用靶向质谱技术检测和定量S9细胞中SULT1A1和1A3/4蛋白。方法:对样品进行胰消化，采用液相色谱-多反应监测质谱法(LC-MRM/MS)对特征肽进行定量分析。用稳定同位素标记(SIL)肽作为内标和定标标准。SULT1A1和SULT1A3/4分别在S9各组分和细胞系样品中进行定量分析。结果:S9和细胞系基质中SULT1A1和SULT1A3/4的相对定量日内和日间变异较低(结论:建立并验证了S9组分和细胞系样品中SULT1A1和SULT1A3/4同时定量的重复性方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Drug metabolism letters Pharmacology, Toxicology and Pharmaceutics-Pharmaceutical Science

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期刊介绍： Drug Metabolism Letters publishes letters and research articles on major advances in all areas of drug metabolism and disposition. The emphasis is on publishing quality papers very rapidly by taking full advantage of the Internet technology both for the submission and review of manuscripts. The journal covers the following areas: In vitro systems including CYP-450; enzyme induction and inhibition; drug-drug interactions and enzyme kinetics; pharmacokinetics, toxicokinetics, species scaling and extrapolations; P-glycoprotein and transport carriers; target organ toxicity and interindividual variability; drug metabolism and disposition studies; extrahepatic metabolism; phase I and phase II metabolism; recent developments for the identification of drug metabolites.