Novel ultra-stable enzymes and methods for proteomics

PROJECT SUMMARY ABSTRACT ! Proteomics is critical for health research as it enables analysis of proteins involved in a broad range of biological processesAlthough proteomics has laid the foundation for many advances in disease diagnosistreatmentand preventionimprovements in sample preparation and processing of protein and sugar biomolecules have lagged far behind impressive developments in mass spectrometryMStechnologiesMost proteomics laboratories are heavily reliant on enzyme technologies that are overyears oldnamely trypsinWhile trypsin has been a very powerful molecular toolthe potential of new classes and capabilities for relevant enzymes have yet to be explored and realizedAt CinderBiowe have developed technologies that yield enzymes with remarkable stability and activity at high temperaturesCand extremes in acidic pHthat also tolerate an unprecedented spectrum of chemicals and solventsThese newly available enzymes hold great promise to rapidly advance many areas of proteomics researchImportantlyoptimal conditions for our enzymatic activities are conditions that denature and solubilize most if not all mesophilic proteinsa current limitation in sample processing for some MS analysesAt CinderBiowe produce a variety of glycohydrolasesincluding sugar de branching enzymesproteasesand other classes of hyper stable enzymes thatfunction optimally at extreme temperatures and pH that assist in exposing target bondsi econditions where mesophilic proteins are denaturedminimally cross react and have little autolysisallowing stable multi enzyme formulationsandare tolerant of many harsh oxidizing reducing chemicalsdetergentsand solventsandhave very fast reaction ratesThese capabilities open possibilities for entirely new methods for proteomic sample preparation for mass spectrometryCinderBio enzyme formulations promise to reduce required sample sizesincrease coveragemake quantitation more reliablesimultaneously de glycosylate and proteolyze samplesand generally streamline upstream sample preparationHere we propose to formulate our enzymes for the explicit purpose of modernizing the proteomic methodologies of sample preparationWe see immediate opportunities to address the growing list of difficult target proteins like histonestransmembrane and glycosylated proteinse gneural tissueswhile increasing digestion efficienciesThese Phase I experiments will focus on establishing proteomics protocols for our enzymes on a set of well known substratesinvestigating simultaneous deglycosylation and proteolysis of a set of glycoproteinsand benchmarking for performance against standard tryptic protocols and enzymes PROJECT NARRATIVE ! Proteomics is critical for health research as it enables analysis of proteins involved in a broad range of biological processesAlthough proteomics has laid the foundation for many advances in disease diagnosistreatmentand preventionimprovements in sample preparation have lagged far behind impressive developments in mass spectrometry technologiesHere we propose to bring an entirely new class of ultrastable enzymes and their capabilities to the forefront of proteomics researchWe think these advances will not only address known protease limitationsbut also expand the scope and effectiveness of proteomic analyses for biological and medical research