Characterization of SH2-Ligand Interactions via LibraryAffinity Selection with Mass Spectrometric Detection
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- 作者：Michele A. Kelly ; Hongbin Liang ; Iou-Iou Sytwu ; Isodoros Vlattas ; Nancy L. Lyons ; Benjamin R. Bowen ; and Lawrence P. Wennogle
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：September 10, 1996
- 年：1996
- 卷：35
- 期：36
- 页码：11747 - 11755
- 全文大小：784K
- 年卷期：v.35,no.36(September 10, 1996)
- ISSN：1520-4995
文摘
Synthetic combinatorial libraries have proven to be a valuablesource of diverse structuresuseful for large-scale biochemical screening. Their use hasgreatly facilitated the study of protein-protein interactions. We have developed a practical technique forscreening such libraries by integratingaffinity chromatography selection with electrospray ionization massspectrometric detection, referred toas library affinity selection-mass spectrometry (LAS-MS). Theprocess allows for rapid and efficientscreening of solution phase libraries and provides detailed informationsuch as the relative affinities ofsubstrates. The method is generally applicable to includenonpeptide libraries; moreover, electrospraytandem mass spectrometry (ES-MS/MS) yields sequence-specificidentification of individual componentswithout the need for chemical tags. This technique is demonstratedusing the Src homology 2 (SH2)domain of phosphatidylinositol 3-kinase (PI 3-kinase). Thecritical importance of methionine in the position+3 (relative to the phosphotyrosine position) is demonstrated in alibrary built with a phosphotyrosinemimic, (phosphonodifluoromethyl)phenylalanine. The describedmethod has broad applicability tocombinatorial library screening.