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首页> 外文期刊>Biochemistry >CHARACTERIZATION OF SH2 - LIGAND INTERACTIONS VIA LIBRARY AFFINITY SELECTION WITH MASS SPECTROMETRIC DETECTION
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CHARACTERIZATION OF SH2 - LIGAND INTERACTIONS VIA LIBRARY AFFINITY SELECTION WITH MASS SPECTROMETRIC DETECTION

机译：通过质谱谱选择法选择亲和力鉴定SH2-配体相互作用。

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摘要

Synthetic combinatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening. Their use has greatly facilitated the study of protein-protein interactions. We have developed a practical technique for screening such libraries by integrating affinity chromatography selection with electrospray ionization mass spectrometric detection, referred to as library affinity selection-mass spectrometry (LAS-MS). The process allows for rapid and efficient screening of solution phase libraries and provides detailed information such as the relative affinities of substrates. The method is generally applicable to include nonpeptide libraries; moreover, electrospray tandem mass spectrometry (ES-MS/MS) yields sequence-specific identification of individual components without the need for chemical tags. This technique is demonstrated using the Src homology 2 (SH2) domain of phosphatidylinositol 3-kinase (PI 3-kinase). The critical importance of methionine in the position +3 (relative to the phosphotyrosine position) is demonstrated in a library built with a phosphotyrosine mimic, (phosphonodifluoromethyl)phenylalanine. The described method has broad applicability to combinatorial library screening.
机译：合成的组合文库已被证明是可用于大规模生化筛选的各种结构的宝贵来源。它们的使用极大地促进了蛋白质间相互作用的研究。我们已经开发了一种通过将亲和色谱选择与电喷雾电离质谱检测相结合来筛选此类文库的实用技术，称为库亲和选择质谱法（LAS-MS）。该方法可以快速有效地筛选溶液相库，并提供详细信息，例如底物的相对亲和力。该方法通常适用于包含非肽文库;此外，电喷雾串联质谱法（ES-MS / MS）无需化学标签即可对单个组分进行序列特异性鉴定。使用磷脂酰肌醇3-激酶（PI 3-激酶）的Src同源2（SH2）域证明了该技术。在用磷酸酪氨酸模拟物（膦二氟甲基）苯丙氨酸构建的文库中证明了蛋氨酸在+3位置（相对于磷酸酪氨酸位置）的至关重要性。所描述的方法在组合文库筛选中具有广泛的适用性。

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