Sachs Aaron N, Pisitkun Trairak, Hoffert Jason D, Yu Ming-Jiun, Knepper Mark A
National Institutes of Health, 10 Center Dr., Bldg. 10, Rm. 6N260, Bethesda, MD 20892-1603, USA.
Am J Physiol Renal Physiol. 2008 Dec;295(6):F1799-806. doi: 10.1152/ajprenal.90510.2008. Epub 2008 Oct 15.
We carried out LC-MS/MS-based proteomic profiling of differential centrifugation fractions from rat inner medullary collecting duct (IMCD): 1) to provide baseline knowledge of the IMCD proteome and 2) to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin-2 (AQP2). IMCD suspensions were freshly prepared from rat kidneys using standard methods. Homogenized samples were subjected to sequential centrifugations at 1,000, 4,000, 17,000, and 200,000 g. These samples, as well as the final supernatant, were subjected to LC-MS/MS analysis. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17,000-g fraction to the 200,000-g fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200,000-g fraction. Immunoblotting for the major phosphorylated forms of AQP2 revealed that phosphorylated AQP2 was present in both the 17,000- and 200,000-g fractions. LC-MS/MS analysis showed that markers of "intracellular vesicles," chiefly endosomal markers, were present in both the 17,000- and the 200,000-g fractions. In contrast, plasma membrane proteins were predominantly present in the 4,000- and 17,000-g fractions. Proteins associated with several multiprotein complexes (e.g., actin-related protein 2/3 complex and proteasome complex) were virtually exclusively present in the 200,000-g fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD database. The data show that both the 17,000- and 200,000-g fractions are highly heterogeneous and cannot be equated with "plasma membrane" and "intracellular vesicle" fractions, respectively, leading us to propose an alternative approach for use of differential centrifugation to assess vesicular trafficking to the plasma membrane.
我们对大鼠肾内髓集合管（IMCD）差速离心分级分离得到的组分进行了基于液相色谱-串联质谱（LC-MS/MS）的蛋白质组分析：1）以提供IMCD蛋白质组的基线知识；2）评估差速离心在评估水通道蛋白-2（AQP2）转运方面的效用。使用标准方法从大鼠肾脏新鲜制备IMCD悬浮液。将匀浆后的样品依次在1000、4000、17000和200000 g下进行离心。这些样品以及最终的上清液都进行了LC-MS/MS分析。初步免疫印迹证实，在加压素类似物dDAVP作用下，17000 g组分中AQP2与200000 g组分中AQP2的比例有所增加，这主要是由于200000 g组分中AQP2减少所致。对AQP2主要磷酸化形式的免疫印迹显示，磷酸化的AQP2存在于17000 g和200000 g组分中。LC-MS/MS分析表明，“细胞内囊泡”的标志物，主要是内体标志物，同时存在于17000 g和200000 g组分中。相比之下，质膜蛋白主要存在于4000 g和17000 g组分中。与几种多蛋白复合物（如肌动蛋白相关蛋白2/3复合物和蛋白酶体复合物）相关的蛋白质实际上仅存在于200000 g组分中。总体而言，我们鉴定出656种蛋白质，其中包括189种先前未出现在IMCD数据库中的蛋白质。数据表明，17000 g和200000 g组分都高度异质，不能分别等同于“质膜”和“细胞内囊泡”组分，这促使我们提出一种使用差速离心评估囊泡向质膜转运的替代方法。
