title: A quantitative proteomics-based competition binding assay to characterize pITAM-protein interactions.
abstract: Characterization of ligand-protein binding is of crucial importance in drug discovery. Classical competition binding assays measure the binding of a labeled ligand in the presence of various concentrations of unlabeled ligand and typically use single purified proteins. Here, we introduce a high-throughput approach to study ligand-protein interactions by coupling competition binding assays with mass spectrometry-based quantitative proteomics. With the use of a phosphorylated immunoreceptor tyrosine-based activation motif (pITAM) peptide as a model, we characterized pITAM-interacting partners in human lymphocytes. The shapes of competition binding curves of various interacting partners constructed in a single set of quantitative proteomics experiments reflect relative affinities for the pITAM peptide. This strategy can provide an efficient approach to distinguish specific interacting partners, including two signaling kinases possessing tandem SH2 domains, SYK and ZAP-70, as well as other SH2 domain-containing proteins such as CSK and PI3K, from contaminants and to measure relative binding affinities of multiple proteins in a single experiment.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, United States.
authors.forename: Lianghai
authors.initials: L
authors.lastname: Hu
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Lipchik
authors.forename: Robert L
authors.initials: RL
authors.lastname: Geahlen
authors.forename: Laurie L
authors.initials: LL
authors.lastname: Parker
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D006801:Humans; D061625:Immunoreceptor Tyrosine-Based Activation Motif; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D010446:Peptide Fragments; D010750:Phosphoproteins