SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR.
SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR.
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SH2-PLA：一种敏感的内解决方法，用于定量通过接近结扎和实时PCR来定量模块化结构域结合。
DOI：
10.1186/s12896-015-0169-1
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发表时间：
2015-06-26
影响因子：
3.5
通讯作者：
Machida K
中科院分区：
文献类型：
--
作者：
Thompson CM;Bloom LR;Ogiue-Ikeda M;Machida K
酪氨酸激酶途径中的磷酸酪氨酸依赖性蛋白质-蛋白质相互作用在细胞功能的许多方面起着至关重要的作用，因此对酪氨酸激酶途径中的磷酸酪氨酸依赖性蛋白质-蛋白质相互作用的研究引起了极大的兴趣。我们以前建立了SH 2分析，磷酸化蛋白质组学方法的基础上膜结合试验，利用纯化的Src同源2（SH 2）结构域作为分子工具来配置文件的全球酪氨酸磷酸化状态的细胞。然而，为了使用这种方法来研究细胞裂解物中特定靶标上的SH 2结合位点，需要消耗大量样品的额外程序，例如下拉或免疫沉淀。我们已经开发了PLA-SH 2，这是一种替代的溶液中模块化结构域结合测定，其利用了邻近连接测定和实时PCR。SH 2-PLA检测试剂盒使用分别识别GST-SH 2探针和细胞EGFR的抗GST和抗EGFR抗体偶联的谷胱甘肽。如果GST-SH 2和EGFR由于SH 2-磷酸酪氨酸相互作用而紧密接近，则将两种寡核苷酸置于合适的距离内以进行连接，从而允许通过实时PCR进行有效的复合物扩增。该试验检测到至少3个数量级的裂解物输入信号，线性范围跨越1-2个数量级，EGFR磷酸酪氨酸的检测限较低。SH 2结合动力学测定PLA-SH 2显示良好的协议与建立远Western分析A431和Cos 1细胞刺激EGF在不同的时间和剂量。此外，我们表明PLA-SH 2可以使用1 μl裂解物而不需要磷酸富集来检测肺癌组织。我们第一次表明，SH 2结构域探针和EGFR在细胞裂解物之间的相互作用，可以确定在微升规模的测定使用SH 2-PLA。该方法的明显益处是低样品要求允许检测难以使用传统蛋白质相互作用测定法分析的样品中的SH 2结合。该特征沿着短的测定运行时间使得该方法成为用于开发高通量测定以确定模块化结构域-配体相互作用的有用平台，其可以在基础和转化癌症研究中具有广泛的应用。本文的在线版本（doi：10.1186/s12896-015-0169-1）包含补充材料，可供授权用户使用。
There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required. We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1–2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment. We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein interaction assays. This feature along with short assay runtime makes this method a useful platform for the development of high throughput assays to determine modular domain–ligand interactions which could have wide-ranging applications in both basic and translational cancer research. The online version of this article (doi:10.1186/s12896-015-0169-1) contains supplementary material, which is available to authorized users.
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影响因子：
--
作者：
Machida, Kazuya;Khenkhar, Malik;Nollau, Peter
通讯作者：
Nollau, Peter
影响因子：
4.2
作者：
Gehring A;He X;Fratamico P;Lee J;Bagi L;Brewster J;Paoli G;He Y;Xie Y;Skinner C;Barnett C;Harris D
通讯作者：
Harris D
影响因子：
3.7
作者：
Machida K;Eschrich S;Li J;Bai Y;Koomen J;Mayer BJ;Haura EB
通讯作者：
Haura EB
影响因子：
14.9
作者：
Hornbeck PV;Kornhauser JM;Tkachev S;Zhang B;Skrzypek E;Murray B;Latham V;Sullivan M
通讯作者：
Sullivan M
影响因子：
46.9
作者：
Fredriksson, S;Gullberg, M;Landegren, U
通讯作者：
Landegren, U