title: High-Throughput Quantification of SH2 Domain-Phosphopeptide Interactions with Cellulose-Peptide Conjugate Microarrays.
abstract: The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define "specificity space," and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment. Here, I describe the fabrication and application of a cellulose-peptide conjugate microarray (CPCMA) platform to the quantitative analysis of SH2 domain specificity space. Included herein are instructions for optimal experimental design with special attention paid to common sources of systematic error, phosphopeptide SPOT synthesis, microarray fabrication, analyte titrations, data capture, and analysis.
authors.affiliation: Department of Human Genetics, The University of Chicago, Chicago, IL, USA. bengelmann9@gmail.com.
authors.forename: Brett W
authors.initials: BW
authors.lastname: Engelmann
mesh_terms: D057166:High-Throughput Screening Assays; D008024:Ligands; D010748:Phosphopeptides / Q000737:chemistry* / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods; D012680:Sensitivity and Specificity; D057188:Workflow; D018909:src Homology Domains
chemical_list: D008024:Ligands; D010748:Phosphopeptides
keywords: Affinity; Context specificity; High-throughput screening; Interactome; Peptide microarray; Phosphotyrosine peptides; Proteomics; Quantitative; SH2 domain; Specificity; Specificity space