Site-Specific Incorporation of Phosphorylation Modifications on the SNAP-Tide High Density Peptide Array

Project Summary Site Specific Incorporation of Phosphorylation Modifications on the SNAP Tide High Density Peptide ArrayPIsMary SOzers and Christopher LWarren Post translational modificationsPTMsincrease the functional complexity and regulation of proteinsand dysregulation of PTMs are implicated in diseaseincluding cancerAlmost all technologies used to detect and study PTMs depend on antibodiestools that are as unreliable as they are ubiquitousAntibody validation from manufacturers is highly variableand antibodiesespecially those directed against PTMssuffer from off target recognitionOur newly developed SNAP Tide arraySpecificity and Affinity for PepTidesis a high densitycustomizableand high throughput peptide microarray platform technologywhich can display up to one million unique peptides encompassing the entire human proteome on a single glass slideThisfold increase in peptide density is possible because of our innovative synthesis processin which peptide coding sequences on a standard DNA microarray are converted into RNA barcoded peptides in vitroand the resulting peptides are addressed back to the arrayPeptide arrays are already used to analyze PTM enzyme specificity because many PTM enzymessuch as those that confer phosphorylationsumoylationand arginine methylationtarget short linear peptide sequences rather than three dimensional protein conformationsHoweverhigh density peptide arrays with PTMs already incorporated are not yet commercially availableIn this proposalwe will innovate upon the SNAP Tide platform to create the first commercially available modified high density peptide arraythe PhosphoSer SNAP Tide arraywhich will display peptides containing phosphoserinepSerone of the most common PTMs in the cellSpecificallywe willCreate a PhosphoSer SNAP Tide array of the whole proteomewith single or multiple phosphoserinespSerper peptide using an orthogonal translation systemOTSwhich allows site specific incorporation of a PTM during translationValidate the presence and localization of pSer peptides on the array using biochemical techniquesincluding mass spectrometryfluorescent labelingantibody detectionand phosphatase treatmentAimAand compare to an existing peptide array modified enzymaticallyAimBandDemonstrate the utility of the PhosphoSer SNAP Tide array by mapping the epitope of antibodies against estrogen receptorat key phosphorylation sites known to play a role in breast cancerAimAand by examining the role of Pincatalyzed peptide bond isomerization in antibody recognition of pSerAimBThis technology allows the site specific incorporation of both phosphorylated and non phosphorylated serine residues at distinct positions within a million peptide sequences on a single arraysomething that cannot be achieved enzymaticallyThe PhosphoSer SNAP Tide array could be employed by manufacturers and end users to check antibody affinitysensitivityoff target bindingspecificityand lot to lot variationBeyond antibody characterizationthe PhosphoSer SNAP Tide technology is anticipated to have a major impact on drug and antigen developmentprotein inhibitorsbiomarker discoveryand diagnostic tools due to its unparalleled customization and affordability Project Narrative Relevance Site Specific Incorporation of Phosphorylation Modifications on the SNAP Tide High Density Peptide ArrayPIsMary SOzers and Christopher LWarren Post translational modificationsespecially phosphorylationare known to be dysregulated during disease processes such as cancerCurrent tools to study protein phosphorylation are plagued by issues of specificity and sensitivityWe propose to develop the PhosphoSerine SNAP Tide technology to examine the specificity and off target binding of antibodies against phosphorylated and non phosphorylated peptides of the human proteome in a high density peptide array formatimproving the value of these crucial tools for therapeuticdiagnosticand research applications