Seeing the unseen: Enhancing purification of labile protein complexes and cells with low surface marker expression

ABSTRACT Asomicsstudies become increasing commonnew methods to identify biomolecule interactions are neededThe ability to isolate specific biomolecules is a fundamental process that spans many biomedical sectors including researchdiagnosticsdrug discoveryand biomarker developmentCommonlysuch isolations are performed by capturing the analyte of interest on a solid structure and then washing away unbound or noninteracting materialThis washing based strategy has important limitations for interactions that are transiently or weakly capturede gprotein complexes involved in post translational modificationProtein protein interactions govern cellular behavior and are consequently transient in natureOnce captureddilutive washing further promotes complex dissociationmaking it difficult to isolate important transient complexesincluding transcriptional machinery and enzyme substrate interactionsYetit is these dynamic regulatory complexes that are appealing as drug targetsdue to the ability to directly modulate cell behavior through their disruptionImproved ability to isolate and analyze weakly bound analytes would be transformative across biomedical disciplinesenabling scientists toseewhat was previously unknownWe will apply a fundamentally new isolation processExclusion based Sample PreparationESPto this important category of analytesESP relies on the passage of magnet bead bound analyte through phase interfaces to achieve purification with much less exposure to pro dissociation factors such as dilutionHereusing a novel model system withtunableaffinitywe will perform a comprehensive characterization of the ESP advantage to drive widespread acceptance and commercialization of this methodAdditionallywe will apply ESP to the capture of weakly bound analytes in animal model tissue samples for the first timevalidating ESP s utility withreal worldsamplesWe will isolate protein complexes that are implicated in the ubiquitous NF kB pathwayusing splenic samples from mice with specific NF kB modulating mutationsSuccessful completion of both Aims will provide a comprehensive quantitative characterization of the technique as well as demonstrating itsdiscoverycapabilitiestogether these will lay the foundation for immediate marketing of ESP into this market as well as futurePhase IIdiscovery basedstudies aimed at mapping unknown branches of theinteractomewith the ultimate goal of discovering new drug targets to benefit human health!