Optimized Methods for Targeted Peptide-Based Quantification of Human Uridine 5′-Diphosphate-Glucuronosyltransferases in Biological Specimens Using Liquid Chromatography-Tandem Mass Spectrometry
Optimized Methods for Targeted Peptide-Based Quantification of Human Uridine 5′-Diphosphate-Glucuronosyltransferases in Biological Specimens Using Liquid Chromatography-Tandem Mass Spectrometry
复制标题
DOI：
10.1124/dmd.113.056291
复制
发表时间：
2014-05-01
影响因子：
3.9
通讯作者：
Usui, Takashi
中科院分区：
文献类型：
--
作者：
Sato, Yuichiro;Nagata, Masanori;Usui, Takashi
本研究的目的是优化人类 13 尿苷 5'-二磷酸-葡萄糖醛酸基转移酶 (UGT) 亚型（UGT1A1、1A3、1A4、1A6、1A7、1A8、1A9、1A10、2B4、2B7、2B10、2B15 和 2B17）的定量方法使用液相色谱-串联质谱法，通过基于肽的靶向定量，对肝、肠和肾微粒体以及表达重组人 UGT 的昆虫细胞膜 (rhUGT) 进行分析。通过组合三种变性剂（尿素、脱氧胆酸钠和辛基葡萄糖苷）和三种变性温度（37℃、60℃和95℃），然后进行胰蛋白酶消化 2-20 小时，比较了目标肽的产生。产生高生产效率的变性条件和消化时间在异构体和样本之间存在显着差异，表明特定优化的重要性。使用最佳方法对每种 UGT 同工型进行定量。 10（1A1、1A3、1A4、1A6、1A9、2B4、2B7、2B10、2B15 和 2B17）、6（1A1、1A3、1A4、1A10、2B7 和 2B17）和 3（1A6、1A9 和 2B7）的表达分别在人类肝脏、肠道和肾脏微粒体中检测到同种型，并且使用多种方案可重复水平。所有亚型均在 rhUGT 中定量。确定人体组织样本和 rhUGT 中的 UGT 水平对于基于体外-体内外推法估计葡萄糖醛酸化对身体清除率的贡献非常重要。
The aim of this study was to optimize methods for quantifying 13 uridine 5 '-diphosphate-glucuronosyltransferase (UGT) isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17) in human liver, intestinal, and kidney microsomes, and in recombinant human UGT-expressing insect cell membranes (rhUGTs) by targeted peptide-based quantification using liquid chromatography-tandem mass spectrometry. Production of targeted peptides was compared by combining three denaturing agents (urea, sodium deoxycholate, and octyl glucoside) and three denaturing temperatures (37 degrees C, 60 degrees C, and 95 degrees C) followed by tryptic digestion for 2-20 hours. Denaturing conditions and digestion times yielding high production efficiency varied markedly among isoforms and specimens, indicating the importance of specific optimization. Each UGT isoform was quantified using the methods found to be optimal. The expression of 10 (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17), 6 (1A1, 1A3, 1A4, 1A10, 2B7, and 2B17), and 3 (1A6, 1A9, and 2B7) isoforms was detected in human liver, intestinal, and kidney microsomes, respectively, and levels were reproducible using multiple protocols. All isoforms were quantified in rhUGTs. Determining the levels of UGTs in human tissue specimens and those in rhUGTs is important for estimating the contribution of glucuronidation to body clearance based on in vitro-in vivo extrapolation.