title: Characterization of SH2-ligand interactions via library affinity selection with mass spectrometric detection.
abstract: 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.
authors.affiliation: CIBA Pharmaceuticals Division, Summit, New Jersey 07901, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Kelly
authors.forename: H
authors.initials: H
authors.lastname: Liang
authors.forename: I I
authors.initials: II
authors.lastname: Sytwu
authors.forename: I
authors.initials: I
authors.lastname: Vlattas
authors.forename: N L
authors.initials: NL
authors.lastname: Lyons
authors.forename: B R
authors.initials: BR
authors.lastname: Bowen
authors.forename: L P
authors.initials: LP
authors.lastname: Wennogle
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D019000:Phosphotyrosine / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D018909:src Homology Domains
chemical_list: D008024:Ligands; D008715:Methionine; D010455:Peptides; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019000:Phosphotyrosine; D011993:Recombinant Fusion Proteins