Development of Functionalized Membranes to Enhance Antibody Sequencing and Screening

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With with award, the Chemical Measurement and Imaging Program in the Division of Chemistry is supporting Professors Merlin Bruening and Xuefei Huang at Michigan State University to create new methods to isolate particular antibodies and determine their sequences. Antibodies are attractive as therapeutic agents due to their specificity, but despite their growing medicinal importance, development and characterization of new antibodies is arduous and expensive. This work aims to simplify antibody development by capturing only the strongest-binding natural antibodies from a mixture and then sequencing these antibodies to enable their synthesis for future investigations or applications. These studies will also develop methods to identify antibody modifications for quality control. The project will involve high school, undergraduate, and graduate students. Additionally, incorporation of research concepts, advanced mass spectrometry, and surface analysis techniques into the undergraduate and particularly graduate curricula is planned. A workshop for high school teachers will focus on protein analysis by MS to further expand the impact of this effort, while internships for minority undergraduate and high school students should contribute to increasing the number of underrepresented students entering the research pipeline.Technically, this project explores membrane-based antibody digestion to control the size of proteolytic peptides and enhance both characterization and sequencing of antibodies using mass spectrometry (MS). Immobilization of proteases such as pepsin in the pores of membranes creates enzymatic reactors, and because protease-containing membranes are only 100 microns thick, high flow rates through membranes give msec digestion times. The research examines whether combining large peptides from msec digestions and small peptides from longer digestions facilitates detection of protein modifications, determination of the location of these modifications, and arrangement of peptides for protein sequencing. Unlike other digestion methods, membrane-based digestion yields long and short peptides with the same enzyme so the molecular masses of short peptides add to give the masses of long peptides. This should lead to a unique and effective method for arranging peptides in a protein sequence based on the relationships between masses of these large and small peptides. Subsequent MS/MS analysis of large and small peptides is expected to give almost complete amino acid coverage for the antibody sequence. Moreover, the large peptides from inexpensive digestion with pepsin-modified membranes will facilitate characterization of antibody oxidation, glycosylation, and deamidation, even in mixtures. Importantly, digestion in pepsin-containing membranes is inexpensive and occurs at low pH to minimize antibody degradation. These studies will also examine whether porous membranes containing antigens can isolate a few tight- or rapid-binding antibodies from a polyclonal mixture. Compared to bead-based methods for isolation, membranes offer lower dead volume, less non-specific adsorption, and finer control of binding times and rinsing to potentially facilitate rapid identification of a few antibodies from a polyclonal mixture. Subsequent sequencing will enable antibody synthesis and application. Thus, the techniques explored in this research may eventually lead to methods for discovering and sequencing antibodies from immunized or infected animals.