Department of Pharmaceutical Sciences, Washington State University, 12 E Spokane Falls Blvd, Spokane, WA 99202, USA.
Department of Pharmaceutical Sciences, Washington State University, 12 E Spokane Falls Blvd, Spokane, WA 99202, USA.
J Pharm Sci. 2021 Jul;110(7):2833-2840. doi: 10.1016/j.xphs.2021.03.020. Epub 2021 Mar 28.
Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are broadly applicable to the characterization of in vitro and in vivo models, in vitro to in vivo extrapolation (IVIVE), and interindividual variability prediction. However, the emerging need of DMET quantification in small sample volumes such as organ-on a chip effluent, organoids, and biopsies requires ultrasensitive protein quantification methods. We present an ultrasensitive method that relies on an optimized sample preparation approach involving acetone precipitation coupled with a microflow-based liquid chromatography-tandem mass spectrometry (µLC-MS/MS) for the DMET quantification using limited sample volume or protein concentration, i.e., liver tissues (1-100 mg), hepatocyte counts (~4000 to 1 million cells), and microsomal protein concentration (0.01-1 mg/ml). The method was applied to quantify DMETs in differential tissue S9 fractions (liver, intestine, kidney, lung, and heart) and cryopreserved human intestinal mucosa (i.e., CHIM). The method successfully quantified >75% of the target DMETs in the trypsin digests of 1 mg tissue homogenate, 15,000 hepatocytes, and 0.06 mg/ml microsomal protein concentration. The precision of DMET quantification measured as the coefficient of variation across different tissue weights, cell counts, or microsomal protein concentration was within 30%. The method confirmed significant extrahepatic abundance of non-cytochrome P450 enzymes such as dihydropyridine dehydrogenase (DPYD), epoxide hydrolases (EPXs), arylacetamide deacetylase (AADAC), paraoxonases (PONs), and glutathione S-transferases (GSTs). The ultrasensitive method developed here is applicable to characterize emerging miniaturized in vitro models and small volume biopsies. In addition, the differential tissue abundance data of the understudied DMETs will be important for physiologically-based pharmacokinetic (PBPK) modeling of drugs.
药物代谢酶和转运体（DMET）的蛋白丰度数据广泛适用于体外和体内模型的特征描述、体外到体内外推（IVIVE）以及个体间变异性预测。然而，在芯片流出物、类器官和活检等小样本量中对 DMET 进行定量的新需求需要超灵敏的蛋白定量方法。我们提出了一种超灵敏的方法，该方法依赖于一种优化的样品制备方法，涉及丙酮沉淀，结合微流控液相色谱-串联质谱（µLC-MS/MS），可用于使用有限的样品量或蛋白浓度，即肝组织（1-100mg）、肝细胞计数（~4000 至 100 万个细胞）和微粒体蛋白浓度（0.01-1mg/ml）对 DMET 进行定量。该方法应用于定量差异组织 S9 级分（肝、肠、肾、肺和心）和冷冻保存的人肠黏膜（即 CHIM）中的 DMET。该方法成功地对 1mg 组织匀浆、15000 个肝细胞和 0.06mg/ml 微粒体蛋白浓度的胰蛋白酶消化物中的 >75%的目标 DMET 进行了定量。DMET 定量的精密度以不同组织重量、细胞计数或微粒体蛋白浓度的变异系数来衡量，其值在 30%以内。该方法证实了非细胞色素 P450 酶如二氢吡啶脱氢酶（DPYD）、环氧化物水解酶（EPXs）、芳基乙酰胺脱乙酰酶（AADAC）、对氧磷酶（PONs）和谷胱甘肽 S-转移酶（GSTs）在肝外的丰度显著增加。这里开发的超灵敏方法适用于描述新兴的小型化体外模型和小体积活检。此外，这些研究较少的 DMET 的差异组织丰度数据对于药物的基于生理的药代动力学（PBPK）建模将是重要的。
