Development of Functionalized Membranes to Enhance Antibody Sequencing and Screening

化学系的化学测量和成像计划正在支持密歇根州立大学的梅林·布鲁宁教授和黄学飞教授创造新的方法来分离特定的抗体并确定它们的序列。由于抗体的特异性，抗体作为治疗药物很有吸引力，但尽管它们在医学上的重要性越来越大，但新抗体的开发和表征是困难和昂贵的。这项工作旨在简化抗体的开发，只从混合物中捕获结合最强的天然抗体，然后对这些抗体进行测序，以便为未来的研究或应用合成它们。这些研究还将开发识别抗体修饰的方法，以进行质量控制。该项目将涉及高中生、本科生和研究生。此外，计划将研究概念、先进的质谱学和表面分析技术纳入本科生课程，特别是研究生课程。一个针对高中教师的研讨会将集中于MS的蛋白质分析，以进一步扩大这一努力的影响，而少数族裔本科生和高中生的实习将有助于增加进入研究渠道的未被充分代表的学生的数量。该项目在技术上探索基于膜的抗体消化，以控制蛋白水解肽的大小，并利用质谱仪(MS)增强抗体的表征和测序。将胃酶等酶固定在膜的孔道中会产生酶反应，由于含有蛋白酶的膜只有100微米厚，因此通过膜的高流速可以提供几毫秒的消化时间。这项研究考察了将来自毫秒级消化的大肽和来自较长消化的小肽组合是否有助于检测蛋白质修饰、确定这些修饰的位置以及用于蛋白质测序的肽的排列。与其他消化方法不同，基于膜的消化产生的长肽和短肽具有相同的酶，所以短肽的分子质量相加得到了长肽的质量。这将导致一种独特而有效的方法，用于根据这些大小多肽之间的质量关系在蛋白质序列中排列多肽。随后对大肽和小肽的MS/MS分析有望给出抗体序列的几乎全部氨基酸覆盖。此外，用胃蛋白酶修饰的膜廉价消化的大肽将有助于抗体氧化、糖基化和脱酰胺的表征，即使在混合物中也是如此。重要的是，在含有胃蛋白酶的膜中消化是廉价的，并且在低pH值下进行，以最大限度地减少抗体的降解。这些研究还将检验含有抗原的多孔膜能否从多克隆混合物中分离出一些紧密或快速结合的抗体。与基于珠粒的分离方法相比，膜提供了更低的死体积，更少的非特异性吸附，以及对结合次数和漂洗的更精细的控制，从而潜在地促进了从多克隆混合物中快速鉴定少数抗体。随后的测序将使抗体的合成和应用成为可能。因此，这项研究中探索的技术最终可能导致从免疫或受感染的动物中发现抗体并对其进行排序的方法。

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.