Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells
Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells
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DOI：
10.1208/s12248-017-0121-5
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发表时间：
2017-09-01
期刊：
影响因子：
4.5
通讯作者：
Unadkat, Jashvant D.
中科院分区：
文献类型：
--
作者：
Kumar, Vineet;Tot Bui Nguyen;Unadkat, Jashvant D.
使用LC-MS/MS的定量蛋白质组学越来越多地用于定量组织和细胞中存在的药物转运蛋白。大多数这些研究通过利用总膜部分而不仅仅是质膜来量化细胞中的总转运蛋白表达。在这里，我们报告的发展和优化的生物素化方法，以量化蛋白质表达的转运蛋白在细胞质膜。Pierce细胞表面分离方案被优化用于质膜分离。将OATP 1B 1表达CHO细胞与0.78 mg/mL膜不可渗透的生物素化试剂（磺基-NHS-SS-生物素）在37 A ℃下孵育1 h，得到质膜的最佳分离。随后，使用LC-MS/MS通过定量蛋白质组学确定质膜中转运蛋白的表达占总转运蛋白的百分比。发现平板接种的表达OATP 1B 1的CHO、MDCK II和HEK 293细胞中OATP 1B 1的平均（+/- SD）质膜表达为79.7%总细胞OATP 1B 1表达的67.7%（+/- 12.2%）和65.3%（+/- 6.8%）。在平板接种的表达OATP 1B 3的HEK 293细胞中OATP 1B 3、平板接种的表达OATP 2B 1的MDCK II细胞中OATP 2B 1和平板接种的表达NTCP的CHO细胞中钠/牛磺胆酸钠共转运多肽（NTCP）的平均（+/- SD）质膜表达为63.2%（+/- 1.6%）、37.1%（+/- 15.7%）和71.7%（+/- 1.2%）。这种定量质膜中转运蛋白表达的方法将在未来用于预测转运蛋白介导的药物处置。
Quantitative proteomics, using LC-MS/MS, is increasingly used to quantify drug transporters present in tissues and cells. Most of these investigations quantify total transporter expression in the cells by utilizing a total membrane fraction, not only the plasma membrane. Here, we report development and optimization of a biotinylation method to quantify protein expression of transporters in the plasma membrane of cells. The Pierce cell surface isolation protocol was optimized for plasma membrane isolation. Incubation of OATP1B1-expressing CHO cells with 0.78 mg/mL of membrane impermeable biotinylation reagent (sulfo-NHS-SS-biotin) at 37A degrees C for 1 h resulted in optimum isolation of the plasma membrane. Subsequently, the expression of transporters in the plasma membrane as a percent of the total was determined by quantitative proteomics using LC-MS/MS. Mean (+/- SD) plasma membrane expression of OATP1B1 in plated OATP1B1-expressing CHO, MDCKII, and HEK293 cells was found to be 79.7% (+/- 4.7%), 67.7% (+/- 12.2%), and 65.3% (+/- 6.8%) of total cell OATP1B1 expression. Mean (+/- SD) plasma membrane expression of OATP1B3 in plated OATP1B3-expressing HEK293 cells, OATP2B1 in plated OATP2B1-expressing MDCKII cells, and sodium/taurocholate co-transporting polypeptide (NTCP) in plated NTCP-expressing CHO cells was 63.2% (+/- 1.6%), 37.1% (+/- 15.7%), and 71.7% (+/- 1.2%), respectively. This method of quantifying transporter protein expression in the plasma membrane will be useful in the future to predict transporter-mediated drug disposition.