Membrane and Monolith Enzyme Reactors for Proteolysis After Capillary Electrophoresis to Enhance Detection of Proteoforms

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With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Merlin Bruening and his group at the University of Notre Dame are working to develop a new method to characterize small changes in the composition of proteins - key elements in the chemistry of life. Subtle differences in composition can have large impacts on protein function (or malfunction) in cells and organisms, even when the protein variants are "coded" by the same gene. A standard approach to this important problem is to digest proteins (break them into smaller, characteristic constituents known as peptides) and then identify them by separation and analysis of the resulting peptides. This can lead to misidentifications (false positives and false negatives), especially when the structural differences are minor and/or the protein sample is complex. The Bruening approach seeks to reduce these misidentifications by preceding digestion with a high-resolution separation of the intact proteins, thereby presenting for simultaneous analysis all the peptides associated with a given protein. The approach requires thorough but rapid digestion - a key challenge and opportunity. In the long run, the insights derived should enhance understanding of phenomena like the development of drug resistance by organisms such as the malaria parasite. Dr. Bruening and his students are sharing insights about this effort at the interface between chemistry and biology through hosting high school students from underrepresented groups in their lab, and through outreach to school teachers.Bottom-up proteomics is a powerful technique for identifying proteins via proteolysis, peptide separation, tandem mass spectrometry (MS/MS), and comparison of mass spectra to the predictions derived from a protein database. However, because this strategy often gives low protein sequence coverage, proteoform identification, which may depend on detecting a specific peptide, is challenging. To address this problem, the Bruening group is developing in-line digestion of proteins separated using capillary zone electrophoresis (CZE). Capillaries containing terminal enzyme-modified membranes or monoliths are being designed to enable digestion just prior to electrospray ionization MS/MS analysis. Thus, the MS/MS spectra of all the proteolytic peptides from a given protein will appear in a narrow time window. Creation of a time-bin criterion will help to decrease both false-positive and false-negative peptide identifications, particularly for proteoforms. Test applications include detection of proteoforms in model systems such as protein mixtures and histone variants.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.