title: The development and application of a quantitative peptide microarray based approach to protein interaction domain specificity space.
abstract: Protein interaction domain (PID) linear peptide motif interactions direct diverse cellular processes in a specific and coordinated fashion. PID specificity, or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs is the basis of this ability. Here, we develop an integrated experimental and computational cellulose peptide conjugate microarray (CPCMA) based approach for the high throughput analysis of PID specificity that provides unprecedented quantitative resolution and reproducibility. As a test system, we quantify the specificity preferences of four Src Homology 2 domains and 124 physiological phosphopeptides to produce a novel quantitative interactome. The quantitative data set covers a broad affinity range, is highly precise, and agrees well with orthogonal biophysical validation, in vivo interactions, and peptide library trained algorithm predictions. In contrast to preceding approaches, the CPCMAs proved capable of confidently assigning interactions into affinity categories, resolving the subtle affinity contributions of residue correlations, and yielded predictive peptide motif affinity matrices. Unique CPCMA enabled modes of systems level analysis reveal a physiological interactome with expected node degree value decreasing as a function of affinity, resulting in minimal high affinity binding overlap between domains; uncover that Src Homology 2 domains bind ligands with a similar average affinity yet strikingly different levels of promiscuity and binding dynamic range; and parse with unprecedented quantitative resolution contextual factors directing specificity. The CPCMA platform promises broad application within the fields of PID specificity, synthetic biology, specificity focused drug design, and network biology.
authors.affiliation: From the ‡The Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637; bengelmann@uchicago.edu.
authors.forename: Brett W
authors.initials: BW
authors.lastname: Engelmann
authors.affiliation: ¶The La Jolla Institute for Allergy and Immunology, La Jolla, California 92037;
authors.forename: Yohan
authors.initials: Y
authors.lastname: Kim
authors.affiliation: ‖The Department of Statistics, The University of Chicago, Chicago, Illinois 60637;
authors.forename: Miaoyan
authors.initials: M
authors.lastname: Wang
authors.affiliation: ¶The La Jolla Institute for Allergy and Immunology, La Jolla, California 92037;
authors.forename: Bjoern
authors.initials: B
authors.lastname: Peters
authors.affiliation: From the ‡The Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637;
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Rock
authors.affiliation: **The Ben May Department for Cancer Research, The University of Chicago, Chicago, Illinois 60637.
authors.forename: Piers D
authors.initials: PD
authors.lastname: Nash
mesh_terms: D000465:Algorithms; D002482:Cellulose / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006001:Glycoconjugates / Q000737:chemistry; D057166:High-Throughput Screening Assays; D019151:Peptide Library; D010748:Phosphopeptides / Q000737:chemistry*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018909:src Homology Domains / Q000235:genetics*
chemical_list: D006001:Glycoconjugates; D019151:Peptide Library; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D002482:Cellulose