Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye
Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye
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DOI：
10.1002/pmic.200300445
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发表时间：
2003-07-01
期刊：
影响因子：
3.4
通讯作者：
Patton, WF
中科院分区：
文献类型：
--
作者：
Martin, K;Steinberg, TH;Patton, WF
对微量磷酸化蛋白和肽的超灵敏检测是揭示哺乳动物系统中许多最重要的信号转导途径的关键要求。蛋白质微阵列是对全局蛋白表达和翻译后修饰（如磷酸化）进行敏感筛选的潜在有用工具。然而，由于缺乏能够方便地检测蛋白激酶靶标的试剂，对信号通路的分析受到阻碍。历史上，磷酸化检测方法依赖于放射性同位素(（γ - p -32)ATP(γ - p -33)ATP标记）或磷氨基酸选择性抗体。这两种方法都有众所周知的缺点。在本研究中，描述了一种小分子荧光团磷光传感器技术，称为Pro-Q钻石染料，它能够超灵敏地对微阵列上显示的肽和蛋白质中的磷酸化氨基酸残基进行全局检测和定量。荧光Pro-Q钻石磷传感器染料技术的效用被证明使用磷蛋白和磷酸肽，以及与蛋白激酶反应在小型化微阵列分析格式进行。代替使用荧光或酶标记的磷酸氨基酸选择性抗体进行检测，一个小的荧光探针被用作磷酸化状态的通用传感器。根据磷酸残基的数量，在各种不同的市售蛋白质阵列底物上的磷酸蛋白检测限为312-625 fg。用Pro-Q金刚石染料表征固定化肽靶标的酶磷酸化，可以很容易地区分特异性和非特异性肽标记，在picogram到亚picogram检测灵敏度水平。
Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((gamma-P-32)ATP(gamma-P-33)ATP labeling) or phosphoamino acid-selective antibodies. Both of these methods suffer from relatively well-known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro-Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro-Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid-selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312-625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro-Q Diamond dye readily permits differentiation between specific and non-specific peptide labeling at picogram to subpicogram levels of detection sensitivity.