Design and directed evolution of an 'Edmanase' enzyme for high-throughput peptide sequencing

PROJECT SUMMARY ABSTRACT SignificanceNext generation sequencingNGShas has transformed the field of genomics and enabled broad adoption of the technology in biologicaltranslationaland most recently clinical researchThe proteomics equivalent to NGS has not yet been realizedAs suchthe goal of this proposal is to develop an innovative and highly scalable digital peptide sequencing platform to effectively scale and democratize protein analysis by providing a simple robust workflow generating massively parallel peptide sequencing informationApproachWe propose to develop a single molecule digital peptide sequencing platformusing a novel enzymatic cyclic degradation schemeparalleling Edman degradation except using mild enzymatic steps rather than harsh chemical reagentsOur assay is comprised of several key technology elementsa population of DNA tagged single molecule peptides on beadsN terminal amino acidNTAAlabeling with PITCEdman chemical agentbinding of NTAA recognition agents labeled with DNA encoding tagswriting of the DNA code to a library element attached to the peptideand enzymatic removal of a single modified NTAA using an engineered Edmanasethe deliverable of this proposalThis assay is cycled for a dozen or more times to generate a DNA library representing peptide sequence information on millions to billions of peptides that is sequenced on a standard NGS instrumentPublic Health ImpactIn summarywe believe our Next Gen peptide sequencing assay usher in the era ofnext gen proteomicseffectively transforming biologicaltranslationaland clinical researchmuch like NGS technology has already done in the genomics space NARRATIVEand OVERALL IMPACTWe propose to develop an innovative and highly scalable digital peptide sequencing platform that reverse translates protein information into DNA library elements sequencable on a standard Next Generation Sequencing instrumentOur assay is democratizing and intrinsically scalable enabling the parallel readout of million to billions of peptides in a single runAs suchwe believe these features will effectively scale and democratize proteomics assays in the researchtranslationalclinicaland drug discovery arenas by providing true next generation protein workflows and instrumentation