Quantitative Transporter Proteomics by Liquid Chromatography with Tandem Mass Spectrometry: Addressing Methodologic Issues of Plasma Membrane Isolation and Expression-Activity Relationship
Quantitative Transporter Proteomics by Liquid Chromatography with Tandem Mass Spectrometry: Addressing Methodologic Issues of Plasma Membrane Isolation and Expression-Activity Relationship
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DOI：
10.1124/dmd.114.061614
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发表时间：
2015-02-01
影响因子：
3.9
通讯作者：
Unadkat, Jashvant D.
中科院分区：
文献类型：
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作者：
Kumar, Vineet;Prasad, Bhagwat;Unadkat, Jashvant D.
为了使用基于生理学的药代动力学模型预测转运蛋白介导的药物处置，一种方法是测量转运活性并将其与细胞系中的蛋白质表达水平（过度表达转运蛋白）相关联，然后通过体外到体内外推（MVE）将其缩放。这种方法做出了两个主要假设。首先，转运蛋白主要在质膜中表达。其次，转运蛋白的表达水平和活性之间存在线性相关性。本研究旨在检验这两个假设。我们评估了两种声称可以将质膜与其他细胞膜分离的市售试剂盒。 Qiagen Qproteome 试剂盒在据称是质膜的部分中产生的蛋白质非常少。 Abcam 相分离试剂盒富集了质膜，但未将其与其他细胞内膜分离。对于 Abcam 方法，从细胞或人肝组织分离的所有亚细胞级分中有机阴离子转运多肽 (OATP) 1B1/2B1 和乳腺癌抗性蛋白 (BCRP) 的表达水平均跟踪 Na+-K+ ATP 酶的表达水平。假设 Na+-K+ ATP 酶主要位于质膜中，这些数据表明测量的转运蛋白也主要位于质膜中。使用短发夹 RNA，我们创建了具有不同程度的 OATP1B1 或 BCRP 表达水平的细胞系克隆。在这些克隆中，OATP1B1 或 BCRP 的转运活性与蛋白质表达水平高度相关 (r(2) > 0.9)。这些数据支持使用来自转运蛋白过表达细胞系的转运蛋白表达水平数据和活性数据来进行转运蛋白介导的药物处置的IVIVE。
To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (over-expressing the transporter) and then scale these to via in vitro to in vivo extrapolation (MVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane. Second, that there is a linear correlation between expression level and activity of the transporter protein. The present study was conducted to test these two assumptions. We evaluated two commercially available kits that claimed to separate plasma membrane from other cell membranes. The Qiagen Qproteome kit yielded very little protein in the fraction purported to be the plasma membrane. The Abcam Phase Separation kit enriched the plasma membrane but did not separate it from other intracellular membranes. For the Abcam method, the expression level of organic anion-transporting polypeptides (OATP) 1B1/2B1 and breast cancer resistance protein (BCRP) proteins in all subcellular fractions isolated from cells or human liver tissue tracked that of Na+-K+ ATPase. Assuming that Na+-K+ ATPase is predominantly located in the plasma membrane, these data suggest that the transporters measured are also primarily located in the plasma membrane. Using short hairpin RNA, we created clones of cell lines with varying degrees of OATP1B1 or BCRP expression level. In these clones, transport activity of OATP1B1 or BCRP was highly correlated with protein expression level (r(2) > 0.9). These data support the use of transporter expression level data and activity data from transporter overexpressing cell lines for IVIVE of transporter-mediated disposition of drugs.