Interindividual Variability in Hepatic Expression of the Multidrug Resistance-Associated Protein 2 (MRP2/ABCC2): Quantification by Liquid Chromatography/Tandem Mass Spectrometry
Interindividual Variability in Hepatic Expression of the Multidrug Resistance-Associated Protein 2 (MRP2/ABCC2): Quantification by Liquid Chromatography/Tandem Mass Spectrometry
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DOI：
10.1124/dmd.111.043810
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发表时间：
2012-05-01
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：
Deo, Anand K.;Prasad, Bhagwat;Unadkat, Jashvant D.
多药相关蛋白2(MRP2)是一种外排转运蛋白，表达于胆小管细胞膜。为了能够在体外在体内外推MRP2对其底物的肝脏处置的贡献，需要关于肝脏MRP2蛋白表达的个体间变异性的数据。因此，我们在华盛顿大学人类肝库(n=51)中使用了先前验证的高效液相色谱/串联质谱仪分析的改进版本来量化MRP2的表达。以MRP2的未标记多肽(LTIIPQDPILFSGSLR)和稳定同位素标记多肽(LTIIPQDPILFSGSL[(C6N1)-C-13-N-15]R)分别作为标准品和内标。从肝组织中分离出膜部分后，进行溶液内胰酶消化。用三种不同的MRP2合成肽浓度添加人血清白蛋白或混合人肝基质(n=51)所创建的质控样品的误差和精密度均小于15%。用替代多肽测定，UW肝库样本中MRP2的平均表达为1.54+/-0.64fmol/µg肝膜蛋白，且与年龄(7-63岁)或性别无关。启动子区域的单核苷酸多态(Rs717620)以前被认为影响MRP2的表达，但并不影响肝脏的MRP2的表达。相反，单核苷酸多态21214G>；A(V417I；rs2273697)与肝脏MRP2表达显著升高相关。
Multidrug-associated protein 2 (MRP2) is an efflux transporter that is expressed at the bile canalicular membrane. To allow in vitro to in vivo extrapolation of the contribution of MRP2 toward hepatic disposition of its substrates, data on the interindividual variability of hepatic MRP2 protein expression are required. Therefore, we quantified the expression of MRP2 in the University of Washington (UW) human liver bank (n = 51) using a modified version of a previously validated liquid chromatography/tandem mass spectrometry assay. An unlabeled (LTIIPQDPILFSGSLR) and stable isotope-labeled (LTIIPQDPILFSGSL[(C6N1)-C-13-N-15]R) surrogate peptide for MRP2 were used as the calibrator and internal standard, respectively. After isolation of the membrane fraction from the liver tissue, in-solution tryptic digestion was conducted. Quality control samples created by spiking human serum albumin or pooled human liver (n = 51) matrix with three different MRP2 synthetic peptide concentrations generated error and precision values of less than 15%. As determined by the surrogate peptide, the average MRP2 expression in the UW liver bank samples was 1.54 +/- 0.64 fmol/mu g liver membrane protein and was found to be independent of age (7-63 years) or sex. A single nucleotide polymorphism in the promoter region (rs717620), previously thought to affect MRP2 expression, did not influence hepatic expression of MRP2. In contrast, the single nucleotide polymorphism 21214G>A (V417I; rs2273697) was associated with significantly higher hepatic MRP2 expression.