Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Am J Physiol Cell Physiol. 2011 Apr;300(4):C755-70. doi: 10.1152/ajpcell.00360.2010. Epub 2011 Jan 5.
Recent advances in mass spectrometry (MS) have provided means for large-scale phosphoproteomic profiling of specific tissues. Here, we report results from large-scale tandem MS [liquid chromatography (LC)-MS/MS]-based phosphoproteomic profiling of biochemically isolated membranes from the renal cortex, with focus on transporters and regulatory proteins. Data sets were filtered (by target-decoy analysis) to limit false-positive identifications to <2%. A total of 7,125 unique nonphosphorylated and 743 unique phosphorylated peptides were identified. Among the phosphopeptides identified were sites on transporter proteins, i.e., solute carrier (Slc, n = 63), ATP-binding cassette (Abc, n = 4), and aquaporin (Aqp, n = 3) family proteins. Database searches reveal that a majority of the phosphorylation sites identified in transporter proteins were previously unreported. Most of the Slc family proteins are apical or basolateral transporters expressed in proximal tubule cells, including proteins known to mediate transport of glucose, amino acids, organic ions, and inorganic ions. In addition, we identified potentially important phosphorylation sites for transport proteins from distal nephron segments, including the bumetanide-sensitive Na-K-2Cl cotransporter (Slc12a1 or NKCC2) at Ser(87), Thr(101), and Ser(126) and the thiazide-sensitive Na-Cl cotransporter (Slc12a3 or NCC) at Ser(71) and Ser(124). A subset of phosphorylation sites in regulatory proteins coincided with known functional motifs, suggesting specific regulatory roles. An online database from this study (http://dir.nhlbi.nih.gov/papers/lkem/rcmpd/) provides a resource for future studies of transporter regulation.
近年来，质谱（MS）技术的发展为特定组织的大规模磷酸化蛋白质组学分析提供了手段。在这里，我们报告了基于大规模串联 MS（液相色谱（LC）-MS/MS）的生物化学分离肾皮质膜的磷酸蛋白质组学分析结果，重点是转运蛋白和调节蛋白。数据集经过（通过靶标-诱饵分析）过滤，将假阳性鉴定限制在<2%。总共鉴定了 7125 个独特的非磷酸化和 743 个独特的磷酸化肽。在鉴定的磷酸肽中，包括转运蛋白的位点，即溶质载体（Slc，n=63）、ATP 结合盒（Abc，n=4）和水通道蛋白（Aqp，n=3）家族蛋白。数据库搜索显示，在转运蛋白中鉴定的大多数磷酸化位点以前未被报道过。大多数 Slc 家族蛋白是表达在近端肾小管细胞中的顶侧或基底外侧转运蛋白，包括已知介导葡萄糖、氨基酸、有机离子和无机离子转运的蛋白。此外，我们还鉴定了来自远曲小管段的转运蛋白的潜在重要磷酸化位点，包括布美他尼敏感的 Na-K-2Cl 共转运蛋白（Slc12a1 或 NKCC2）在 Ser(87)、Thr(101)和 Ser(126)和噻嗪敏感的 Na-Cl 共转运蛋白（Slc12a3 或 NCC）在 Ser(71)和 Ser(124)。调节蛋白中的一部分磷酸化位点与已知的功能基序吻合，表明具有特定的调节作用。本研究的在线数据库（http://dir.nhlbi.nih.gov/papers/lkem/rcmpd/）为转运蛋白调节的未来研究提供了资源。
