Differential Detergent Fractionation of Membrane Protein From Small Samples of Hepatocytes and Liver Tissue for Quantitative Proteomic Analysis of Drug Metabolizing Enzymes and Transporters.
Differential Detergent Fractionation of Membrane Protein From Small Samples of Hepatocytes and Liver Tissue for Quantitative Proteomic Analysis of Drug Metabolizing Enzymes and Transporters.
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DOI：
10.1016/j.xphs.2020.10.037
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发表时间：
2021-01
影响因子：
3.8
通讯作者：
Smith PC
中科院分区：
文献类型：
--
作者：
Qasem RJ;Fallon JK;Nautiyal M;Mosedale M;Smith PC
从有限的样品中分离足够的膜蛋白对基于ms的药物代谢酶（DMEs）和转运体的定量靶向绝对蛋白质组学（QTAP）具有挑战性。本研究通过QTAP定量选择DMEs和转运体，评估了从逐渐减少的小鼠原代肝细胞（500万至5万个细胞）和有限肝组织（25至50毫克）中分离膜蛋白的差异洗涤剂分离（DDF）。用洋地黄苷和Triton-X-100两种非离子型洗涤剂依次渗透细胞和提取膜蛋白。与膜蛋白提取试剂盒、低渗缓冲液匀浆和差速离心（DC）进行比较。随着肝细胞数量的增加，DDF的膜蛋白产量呈线性增加，并且通过更高的细胞质与膜蛋白产量之比可以证明其具有更好的通透性。DDF产生的肝组织膜蛋白是DC的5倍。DMEs和转运体的浓度在DDF从逐渐减少的肝细胞中制备的组分中保持一致，但在kit组分中下降。在肝组织中，DDF的浓度相对于kit和DC较高。综上所述，从有限样品中对膜蛋白进行顺序地黄皂苷和Triton-X-100分离是一种高效、可重复和经济的方法，可用于DMEs和转运体的QTAP。
The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing enzymes (DMEs) and transporters. This study evaluated differential detergent fractionation (DDF) of membrane protein from progressively smaller numbers of primary mouse hepatocytes (5 million down to 50,000 cells) and limited liver tissue (25 to 50 mg) in quantifying select DMEs and transporters by QTAP. Two non-ionic detergents, digitonin and Triton-X-100, were applied in sequence to permeabilize cells and extract membrane proteins. Comparison was made with a membrane protein extraction kit and with homogenization in hypotonic buffer and subsequent differential centrifugation (DC). DDF produced linear membrane protein yields with increasing hepatocyte numbers and better permeabilization evidenced by the higher ratio of cytosolic to membrane protein yields. DDF produced 5-times more membrane protein from liver tissue than DC. The concentration of DMEs and transporters remained consistent in the fractions prepared by DDF from progressively smaller numbers of hepatocytes, but declined in kit fractions. In liver tissue, the concentrations were comparatively higher in DDF versus kit and DC. In conclusion, sequential digitonin and Triton-X-100 fractionation of membrane protein from limited samples is efficient, reproducible and cost-effective for QTAP of DMEs and transporters.
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影响因子：
4.1
作者：
STRANCAR, A;RASPOR, P;JOSIC, D
通讯作者：
JOSIC, D
影响因子：
3.9
作者：
Higgins, J. William;Bao, Jing Q.;Zamek-Gliszczynski, Maciej J.
通讯作者：
Zamek-Gliszczynski, Maciej J.
影响因子：
4.4
作者：
Fallon, John K.;Neubert, Hendrik;Smith, Philip C.
通讯作者：
Smith, Philip C.
影响因子：
4.4
作者：
Chang, Ying-Hua;Gregorich, Zachery R.;Chen, Albert J.;Hwang, Leekyoung;Guner, Huseyin;Yu, Deyang;Zhang, Jianyi;Ge, Ying
通讯作者：
Ge, Ying
影响因子：
4.2
作者：
Feroz, Hasin;Kwon, HyeYoung;Kumar, Manish
通讯作者：
Kumar, Manish