Centre for Applied Pharmacokinetic Research, Division of Pharmacy and Optometry, University of Manchester, Manchester, United Kingdom (B.A., Z.M.A.-M., A.R.-H., J.B.); Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Inc., Groton, Connecticut (A.D., M.N., J.J.N., T.C.G.); and Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, United Kingdom (A.R.-H.).
Centre for Applied Pharmacokinetic Research, Division of Pharmacy and Optometry, University of Manchester, Manchester, United Kingdom (B.A., Z.M.A.-M., A.R.-H., J.B.); Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Inc., Groton, Connecticut (A.D., M.N., J.J.N., T.C.G.); and Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, United Kingdom (A.R.-H.)
Drug Metab Dispos. 2018 Jun;46(6):805-812. doi: 10.1124/dmd.117.079475. Epub 2018 Mar 26.
Quantitative proteomic methods require optimization at several stages, including sample preparation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and data analysis, with the final analysis stage being less widely appreciated by end-users. Previously reported measurement of eight uridine-5'-diphospho-glucuronosyltransferases (UGT) generated by two laboratories [using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT)] reflected significant disparity between proteomic methods. Initial analysis of QconCAT data showed lack of correlation with catalytic activity for several UGTs (1A4, 1A6, 1A9, 2B15) and moderate correlations for UGTs 1A1, 1A3, and 2B7 (s = 0.40-0.79, < 0.05; = 0.30); good correlations were demonstrated between cytochrome P450 activities and abundances measured in the same experiments. Consequently, a systematic review of data analysis, starting from unprocessed LC-MS/MS data, was undertaken, with the aim of improving accuracy, defined by correlation against activity. Three main criteria were found to be important: choice of monitored peptides and fragments, correction for isotope-label incorporation, and abundance normalization using fractional protein mass. Upon optimization, abundance-activity correlations improved significantly for six UGTs (s = 0.53-0.87, < 0.01; = 0.48-0.73); UGT1A9 showed moderate correlation (s = 0.47, = 0.02; = 0.34). No spurious abundance-activity relationships were identified. However, methods remained suboptimal for UGT1A3 and UGT1A9; here hydrophobicity of standard peptides is believed to be limiting. This commentary provides a detailed data analysis strategy and indicates, using examples, the significance of systematic data processing following acquisition. The proposed strategy offers significant improvement on existing guidelines applicable to clinically relevant proteins quantified using QconCAT.
定量蛋白质组学方法需要在几个阶段进行优化，包括样品制备、液相色谱-串联质谱（LC-MS/MS）和数据分析，而最终的分析阶段则较少受到终端用户的关注。此前，两个实验室报告的 8 种尿苷-5'-二磷酸葡糖醛酸基转移酶（UGT）的测量结果[使用稳定同位素标记（SIL）肽或定量串联体（QconCAT）]反映了蛋白质组学方法之间存在显著差异。对 QconCAT 数据的初步分析表明，对于几种 UGT（1A4、1A6、1A9、2B15），其与催化活性缺乏相关性，而对于 UGTs 1A1、1A3 和 2B7 则具有中等相关性（s=0.40-0.79，<0.05；=0.30）；在相同实验中，细胞色素 P450 活性与丰度的测量之间显示出良好的相关性。因此，从未处理的 LC-MS/MS 数据开始，进行了系统的数据分析综述，旨在提高准确性，通过与活性的相关性来定义。发现有三个主要标准很重要：监测肽和片段的选择、同位素标记掺入的校正以及使用分数蛋白质量进行丰度归一化。经过优化，对于 6 种 UGT（s=0.53-0.87，<0.01；=0.48-0.73），丰度-活性相关性显著提高；UGT1A9 显示出中等相关性（s=0.47，=0.02；=0.34）。没有发现虚假的丰度-活性关系。然而，对于 UGT1A3 和 UGT1A9，方法仍然不够理想；这里认为标准肽的疏水性是有限制的。本评论提供了详细的数据分析策略，并通过示例指出了在获取后进行系统数据处理的重要性。所提出的策略为使用 QconCAT 定量的临床相关蛋白提供了显著优于现有指南的改进。
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