Protein ProFiler Microfluidic Electrophoresis System for Proteomics

AbstractThe current estimate of the number of human genes is aboutin contrast to the number of presumed proteinswhich likely exceeds one millionThis increased complexity is the result of post transcriptional and post translational modificationsPTMsoccurring at multiple sites and in a quantitative variable mannerThe overwhelming result is a combinatorially expanding complexity of proteoforms that is a challenge to proteomic studies in general and the study of biological mechanisms in particularAmong the new tools utilized in this efforttop down mass spectrometryTD MSanalyzes intact proteins with capable mass spectrometersand in doing sois ideally suited towards preserving the complexity of multiply modified proteinsHoweverbecause the larger intact proteins acquire multiple charged states in the MSpeak capacity is significantly limitedAs a consequencedeep mining of the proteome by TD MS is extraordinarily reliant on highly resolving protein separations and quantitative recoveryMost separation systems historically utilized for conventional proteomic mass spectrometry rely on separation systems ideally suited for peptidenot intact proteinsHence there is high demand for efficientrapidand quantitative separations systemsspecially designed for intact proteins and TD MSThe lack of such separations technology is a major roadblock to TD MS progress and its wider acceptanceWe have developed a separations device that utilizes a specially designed and patented microfluidic glass plate with proprietary coatings that achieves high resolution separations of intact proteins based on truly orthogonal protein propertiessize and chargeWhile similar in principle to conventionalD gel electrophoresisour microfluidic Protein ProFileravoids rigid gelsMSincompatible denaturing detergentse gSDSand accomplishes separations by sizefollowed by isoelectric focusing to achieve the highest sensitivityCollection of proteins is accomplished by selective or non selective meanspermitting tailoring the population of collected proteins to the goals of the experiment and MS capabilitiesPreviously we have demonstrated high performance metrics of the system one dimension at a timeHoweverthis project is focused on completion of the quantitative protein recovery subsystemencompassed in one specific aimSuccessful achievement of the milestone underlying the specific aim and ultimate commercialization will not only demonstrate intact protein separations with quantitative recovery in an automatedMS compatibleliquid based microfluidic platformbut will play an impactfulenabling role for the TD MS proteomics communityeffectively providing the necessary tools to decipher the PTM challenges of modern biology Project Narrative This project will complete development of the final subsystemprotein collection subsystemto allow integration into the larger much needed separations technology for top down proteomicscalled the Protein ProFilerThe ProFiler is a microfluidicself contained two dimensional electrophoresis deviceseparating proteins by size using an flowable liquid polymer and isoelectric point using covalently bound pH buffersThe subsystem will recover separated intact proteins in a format compatible with a top down proteomics approachthereby enhancing the acceptance and enabling ultimate realization of the promise of proteomics and its application to precision medicine