Traceless immobilization and SAMDI mass spectrometry enables high-throughput screening of any analyte.

High throughput screeningHTSis one of the most powerful unbiased approaches to identify small molecule modulators of distinct biochemical activitiesFor thisassays are developed for an enzyme of interest and up to millions of compounds are screened to identify novel drug leadsMost HTS assays rely on labels fluorescent or chemical as a readout for activityLabels are now known to introduce artifacts and false positives that slow down progress and drive up costsMoreovernot all enzymes are amenable to label based approachescontributing to the lack of drugs for many clinically relevant targetsThis proposal offers a universal solution by developing a novel label free technology that could be applied to screen any biochemical activity using native substratesThe approach uses self assembled monolayersSAMsof alkanethiolates on gold presenting atrifluoromethylphenyl diazirineTPDgroup that upon irradiation generates a reactive carbene that inserts into a broad range of chemical bonds to covalently immobilize the analyte of interestA key advantage of using SAMs on gold is their compatibility with matrix assisted laser desorption ionizationMALDImass spectrometryMSthat provides a label free and rapidquantitative readout of immobilized analytesThe techniquetermed traceless immobilizationTISAMDI offers a novelpowerful tool that makes all enzymesscreenableopening new therapeutic avenues for challenging and previously intractable targets Project Narrative This proposal addresses the lack of label free and high throughput assays amenable for drug discovery applicationsThe project aims to develop a novel traceless immobilization chemistry that utilizes self assembled monolayers of alkanethiolates on gold presenting atrifluoromethylphenyl diazirineTPDgroup that upon irradiation generates a reactive carbene that inserts into a broad range of chemical bonds to covalently immobilize the analyte of interestWe will demonstrate proof of concept by developing the first label free assay of Pactivity and perform a high throughput screen to identify novel inhibitorsNotablythe approach is flexible and can be used to measure virtually any biochemical activityopening avenues to assay challenging and previously intractable targets