UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; Department of Pharmaceutical Sciences, College of Pharmacy, King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), King Abdullah International Medical Research Center (KAIMRC), National Guard Health Affairs, Riyadh, Saudi Arabia.
UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Pharm Sci. 2021 Jan;110(1):87-96. doi: 10.1016/j.xphs.2020.10.037. Epub 2020 Oct 22.
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 (25-50 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.
从有限的样本中分离足够的膜蛋白对于基于 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）是具有挑战性的。本研究通过 QTAP 评估了从逐渐减少的数量的原代小鼠肝细胞（500 万至 50,000 个细胞）和有限的肝组织（25-50mg）中差异去污剂分级（DDF）分离膜蛋白，以定量选择的 DMEs 和转运体。两种非离子型去污剂，皂素和 Triton-X-100，依次用于细胞通透和提取膜蛋白。与膜蛋白提取试剂盒以及低渗缓冲液的匀浆和随后的差速离心（DC）进行了比较。DDF 产生了线性的膜蛋白产量，随着肝细胞数量的增加而增加，并且通过更高的胞质蛋白与膜蛋白产量比证明了更好的通透性。DDF 从肝组织中产生的膜蛋白比 DC 多 5 倍。从数量逐渐减少的肝细胞中通过 DDF 制备的分数中 DMEs 和转运体的浓度保持一致，但在试剂盒分数中下降。在肝组织中，DDF 与试剂盒和 DC 相比，浓度相对较高。总之，从有限的样本中顺序用皂素和 Triton-X-100 分级分离膜蛋白对于 DMEs 和转运体的 QTAP 是高效，可重复且具有成本效益的。
