Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (J.K.F., P.C.S.); and Department. of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Inc., Andover, Massachusetts, (H.N.) and Groton, Connecticut, (T.C.G.).
Drug Metab Dispos. 2013 Dec;41(12):2076-80. doi: 10.1124/dmd.113.053801. Epub 2013 Sep 17.
Quantification methods employing stable isotope-labeled peptide standards and liquid chromatography-tandem mass spectrometry are increasingly being used to measure enzyme amounts in biologic samples. Isoform concentrations, combined with catalytic information, can be used in absorption, distribution, metabolism, and excretion studies to improve accuracy of in vitro/in vivo predictions. We quantified isoforms of uridine-diphosphate glucuronosyltransferase (UGT) 1A and 2B in 12 commercially available recombinant UGTs (recUGTs) (n = 49 samples) using nano-ultra-high-performance liquid chromatography-tandem mass spectrometry with selected reaction monitoring). Samples were trypsin-digested and analyzed using our previously published method. Two MRMs were collected per peptide and averaged. Where available, at least two peptides were measured per UGT isoform. The assay could detect UGTs in all recombinant preparations: recUGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17, with limit of detection below 1.0 pmol/mg protein for all isoforms. The assay had excellent linearity in the range observed (2-15.5 pmol/mg, after dilution). Examples of concentrations determined were 1465, 537, 538, 944, 865, 698, 604, 791, 382, 1149, 307, and 740 pmol/mg protein for the respective isoforms. There was a 6.9-fold difference between the maximum and minimum recUGT concentrations. The range of concentrations determined indicates that catalytic rates per mg total protein in vitro will not accurately reflect isoform inherent specific activity for a particular drug candidate. This is the first report of a targeted precise quantification of commercially available recUGTs. The assay has potential for use in comparing UGT amounts with catalytic activity determined using probe substrates, thus allowing representation of catalysis as per pmol of UGT isoform.
采用稳定同位素标记肽标准品和液相色谱-串联质谱的定量方法越来越多地用于测量生物样品中的酶含量。同工型浓度，结合催化信息，可用于吸收、分布、代谢和排泄研究，以提高体外/体内预测的准确性。我们使用带有选择反应监测的纳超高效液相色谱-串联质谱法，对 12 种市售重组 UGT（recUGT）（n=49 个样本）中的尿苷二磷酸葡萄糖醛酸基转移酶（UGT）1A 和 2B 同工型进行了定量。样品用胰蛋白酶消化，并使用我们之前发表的方法进行分析。每个肽段采集两个 MRM 并取平均值。对于每个 UGT 同工型，至少测量两个肽段。该测定法可检测所有重组制剂中的 UGT：recUGT1A1、1A3、1A4、1A6、1A7、1A8、1A9、1A10、2B4、2B7、2B15 和 2B17，所有同工型的检测限均低于 1.0 pmol/mg 蛋白。该测定法在观察到的范围内（2-15.5 pmol/mg，稀释后）具有极好的线性。所确定的浓度示例分别为相应同工型的 1465、537、538、944、865、698、604、791、382、1149、307 和 740 pmol/mg 蛋白。最大和最小 recUGT 浓度之间存在 6.9 倍的差异。所确定的浓度范围表明，体外每毫克总蛋白的催化速率不会准确反映特定候选药物的同工型固有比活性。这是首次报道市售 recUGT 的靶向精确定量。该测定法具有与使用探针底物确定的催化活性进行比较的潜力，从而可以根据 UGT 同工型的 pmol 表示催化作用。
