Multiplex and Label-Free Relative Quantification Approach for Studying Protein Abundance of Drug Metabolizing Enzymes in Human Liver Microsomes Using SWATH-MS

We describe a sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based method for label-free, simultaneous, relative quantification of drug metabolism enzymes in human liver microsomes (HLM; n = 78). In-solution tryptic digestion was aided by a pressure cycling method, which allowed a 90 min incubation time, a significant reduction over classical protocols (12–18 h). Digested peptides were separated on an Acquity UHPLC Peptide BEH C18 column using a 60 min gradient method at a flow rate of 0.100 mL/min. The quadrupole-time-of-flight mass spectrometer (ESI-QTOFMS) was operated in positive electrospray ionization mode, and data were acquired by data-dependent acquisition (DDA) and SWATH-MSALL mode. A pooled HLM sample was used as a quality control to evaluate variability in digestion and quantification among different batches, and inter-batch %CV for various proteins was between 3.1 and 7.8%. Spectral library generated from the DDA data identified 1855 distinct proteins and 25 681 distinct peptides at a 1% global false discovery rate (FDR). SWATH data were queried and analyzed for 10 major cytochrome P450 (CYP) enzymes using Skyline, a targeted data extraction software. Further, correlation analysis was performed between functional activity, protein, and mRNA expression for ten CYP enzymes. Pearson correlation coefficient (r) between protein and activity for CYPs ranged from 0.314 (CYP2C19) to 0.767 (CYP2A6). A strong correlation was found between CYP3A4 and CYP3A5 abundance and activity determined using midazolam and testosterone (r > 0.600, p r > 0.400–0.600, p r p < 0.05) for CYP2C8, CYP2C19, and CYP2D6. The findings suggest the suitability of SWATH-MS based method as a valuable and relatively fast analytical technique for relative quantification of proteins in complex biological samples. We also show that protein abundance is a better surrogate than mRNA to predict the activity of CYP activity.我们报道了一种基于全理论碎片离子质谱连续窗口采集（SWATH-MS）的无标记（label-free）定量方法，可同时对人肝微粒体（HLM; n = 78）中的药物代谢酶进行相对定量分析。采用压力循环法辅助溶液内胰蛋白酶消化，将孵育时长缩短至90分钟，较经典实验方案的12~18小时大幅缩减。消化得到的肽段经Acquity超高效液相色谱（UHPLC）肽段BEH C18色谱柱分离，采用60分钟梯度洗脱程序，流速设置为0.100 mL/min。本研究使用正电喷雾电离模式运行四极杆-飞行时间质谱仪（ESI-QTOFMS），并通过数据依赖性采集（DDA）与SWATH-MSALL模式采集质谱数据。以混合人肝微粒体样品作为质控样本，评估不同批次间消化与定量流程的重复性，各目标蛋白的批间变异系数（%CV）介于3.1%~7.8%之间。基于DDA数据构建的谱库，在全局错误发现率（FDR）为1%的条件下，共鉴定出1855种独特蛋白质与25681条独特肽段。利用目标数据提取软件Skyline，针对10种主要细胞色素P450（CYP）酶对SWATH数据进行检索与分析。此外，我们针对10种CYP酶的功能活性、蛋白质丰度与信使RNA（mRNA）表达水平开展了相关性分析。CYP酶的蛋白质丰度与活性间的皮尔逊相关系数（r）范围为0.314（CYP2C19）至0.767（CYP2A6）。其中，CYP3A4与CYP3A5的丰度与通过咪达唑仑和睾酮测定的酶活性之间呈现强相关性（r > 0.600, p < 0.05）；而CYP2C8、CYP2C19及CYP2D6则呈现中等强度相关性（r为0.400~0.600, p < 0.05）。本研究结果表明，基于SWATH-MS的分析方法可作为一种高效且相对快速的分析技术，适用于复杂生物样品中蛋白质的相对定量研究。同时我们证实，相较于mRNA表达，蛋白质丰度是预测CYP酶活性的更佳替代指标。