Phos-tag 是一种功能性分子,可选择性地捕获中性水溶液中的磷酸单酯双阴离子。Phos-tag 对磷酸单酯双阴离子的亲和力是生物体中存在的其他阴离子(例如羧酸阴离子)的 10,000 倍以上。我们已经开发并应用了基于 Phos-tag 的有用的磷酸化蛋白质组学技术。这篇综述描述了 Phos-tag 的发展历史,并概述了已投入实际应用的三种主要技术。第一种是使用带有亲水层析载体(Phos-tag 聚合物珠)的 Phos-tag 衍生物分离和浓缩磷酸肽和磷蛋白的技术。第二种是使用 Phos-tag 生物素检测各种阵列上的磷酸肽和磷酸蛋白的技术。第三种是使用 Phos-tag 丙烯酰胺通过电泳分离和检测磷蛋白的技术。我们希望这三项技术能为磷酸蛋白质组学,并最终为生命科学研究做出重大贡献。
意义
作者发现 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato 的双核金属配合物在接近生理条件。在两个金属离子上具有空位的金属络合物适用于作为桥接配体的磷酸单酯双阴离子( R- OPO 3 2- )的接入。双核锌 (II) 复合物 (Zn 2+ –Phos-tag) 在中性 pH 值下与对硝基苯基磷酸二价阴离子 ( K d = 2.5 × 10 -8 M)强烈结合。对 SO 4 2- , CH 3 COO - , Cl的阴离子选择性指标− , 25 °C 时磷酸双苯酯单阴离子为 5.2 × 10 3 , 1.6 × 10 4 , 8.0 × 10 5 , > 2 × 10 6, 分别。我们一直参与开发技术,使用 Phos-tag 分子及其衍生物来分析磷酸化的生物分子。迄今为止,Phos-tag 技术已为磷酸蛋白质组学的多种程序的开发做出了贡献,包括用于分离和富集磷酸肽和磷蛋白的磷酸亲和色谱技术,用于检测蛋白质的各种微阵列/芯片技术磷酸化和磷酸亲和电泳技术,用于检测磷蛋白迁移率的变化。在这篇评论文章中,作者介绍了基于 Phos-tag 的技术进步对磷酸化蛋白质组学的影响。
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History of Phos-tag technology for phosphoproteomics
Phos-tag is a functional molecule that selectively captures a phosphate monoester dianion in neutral aqueous solutions. The affinity of Phos-tag for phosphate monoester dianions is more than 10,000 times greater than that for other anions present in living organisms, such as carboxylic acid anions. We have developed and applied useful techniques for phosphoproteomics based on Phos-tag. This review describes the history of Phos-tag development and outlines three main technologies that have been put to practical use. The first is a technique to separate and concentrate phosphopeptides and phosphoproteins using a Phos-tag derivative with a hydrophilic chromatography carrier (Phos-tag polymer beads). The second is a technology to detect phosphopeptides and phosphoproteins on various arrays using Phos-tag biotin. The third is a technique to separate and detect phosphoproteins by electrophoresis using Phos-tag acrylamide. We hope that these three technologies will make a significant contribution to phosphoproteomics and, ultimately, to life science research.
Significance
The authors found that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato acted as a novel phosphate-binding tag nanomolecule, Phos-tag, in an aqueous solution under near physiological conditions. The metal complex having a vacancy on two metal ions is suitable for the access of a phosphomonoester dianion (R-OPO32−) as a bridging ligand. A dinuclear zinc(II) complex (Zn2+–Phos-tag) strongly binds to a p-nitrophenyl phosphate dianion (Kd = 2.5 × 10−8 M) at a neutral pH. The anion selectivity indexes against SO42−, CH3COO−, Cl−, and the bisphenyl phosphate monoanion at 25 °C are 5.2 × 103, 1.6 × 104, 8.0 × 105, and > 2 × 106, respectively. We have been involved in developing technologies by using the Phos-tag molecule and its derivatives to permit the analysis of phosphorylated biomolecules. To date, Phos-tag technology has contributed to the development of several procedures for phosphoproteomics, including a phosphate-affinity chromatography technique for the separation and enrichment of phosphopeptides and phosphoproteins, a wide variety of microarray/on-chip techniques for the detection of protein phosphorylation, and a phosphate-affinity electrophoresis technique for the detection of shifts in the mobilities of phosphoproteins. In this review article, the authors introduce the impact of Phos-tag-based technological advances for phosphoproteomics.