Qu Na, Wan Bin, Guo Liang-Hong
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085, China.
Analyst. 2008 Sep;133(9):1246-9. doi: 10.1039/b807133k. Epub 2008 Jul 28.
Protein phosphorylation plays an important role in many significant cellular processes, and has thus gained tremendous interest in the field of proteomics. The electro-active tyrosine residue, as an important receptor of phosphorylation in proteins, exhibits electro-inactivity after being phosphorylated on the hydroxy group of its aromatic ring. In this study, the electrochemical oxidation of tyrosine on indium tin oxide (ITO) electrodes was catalyzed with an electron mediator Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine) and was employed as a signal reporter to differentially detect non-phosphorylated and phosphorylated peptides. A short, tyrosine-containing peptide glu-glu-glu-glu-glu-tyr (EY-6) was immobilized on an ITO surface using the layer-by-layer self-assembly method, and was detected by cyclic voltammetry in an Os(bpy)(3)(2+) solution. The limit of detection was about 0.23 microg mL(-1) EY-6 in solution. The phosphorylated peptide glu-glu-glu-glu-glu-tyr-OP (EY-6P) did not produce an appreciable oxidation current on the electrode. Surface plasmon resonance measurements revealed that the amount of EY-6 and EY-6P adsorbed on the sensor chip surface was 269 and 378 pg mm(-2), respectively. The poly(glu, tyr) (4 : 1) peptide, a protein tyrosine kinase substrate, was also detected by the same approach, with a detection limit of 0.65 microg mL(-1). This new approach offers the possibility of label-free and on-chip detection of protein tyrosine kinase activity.
蛋白质磷酸化在许多重要的细胞过程中发挥着重要作用，因此在蛋白质组学领域引起了极大的关注。电活性酪氨酸残基作为蛋白质中磷酸化的重要受体，在其芳香环的羟基被磷酸化后表现出电惰性。在本研究中，铟锡氧化物（ITO）电极上酪氨酸的电化学氧化由电子媒介体Os(bpy)(3)(2+)（bpy = 2,2'-联吡啶）催化，并用作信号报告物以差异检测非磷酸化和磷酸化的肽。使用层层自组装方法将短的含酪氨酸肽glu-glu-glu-glu-glu-tyr（EY-6）固定在ITO表面，并在Os(bpy)(3)(2+)溶液中通过循环伏安法进行检测。溶液中EY-6的检测限约为0.23μg mL(-1)。磷酸化肽glu-glu-glu-glu-glu-tyr-OP（EY-6P）在电极上没有产生明显的氧化电流。表面等离子体共振测量表明，吸附在传感器芯片表面的EY-6和EY-6P的量分别为269和378 pg mm(-2)。聚（glu，tyr）（4:1）肽，一种蛋白质酪氨酸激酶底物，也通过相同的方法进行检测，检测限为0.65μg mL(-1)。这种新方法为蛋白质酪氨酸激酶活性的无标记和芯片上检测提供了可能性。
