CHARACTERIZATION OF SH2 - LIGAND INTERACTIONS VIA LIBRARY AFFINITY SELECTION WITH MASS-SPECTROMETRIC DETECTION

Synthetic combinatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening. Th eir use has greatly facilitated the study of protein-protein interacti ons. We have developed a practical technique for screening such librar ies by integrating affinity chromatography selection with electrospray ionization mass spectrometric detection, referred to as library affin ity selection-mass spectrometry (LAS-MS). The process allows for rapid and efficient screening of solution phase libraries and provides deta iled information such as the relative affinities of substrates. The me thod is generally applicable to include nonpeptide libraries; moreover , electrospray tandem mass spectrometry (ES-MS/MS) yields sequence-spe cific identification of individual components without the need for che mical tags. This technique is demonstrated using the Src homology 2 (S H2) domain of phosphatidylinositol 3-kinase (PI 3-kinase). The critica l importance of methionine in the position +3 (relative to the phospho tyrosine position) is demonstrated in a library built with a phosphoty rosine mimic, (phosphonodifluoromethyl)phenylalanine. The described me thod has broad applicability to combinatorial library screening.