Development of Functionalized Membranes to Enhance Antibody Sequencing and Screening

开发功能化膜以增强抗体测序和筛选

• 批准号: 1506315
• 负责人: Merlin Bruening
• 金额: $ 45万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1506315&HistoricalAwards=false
• 关键词: Development; Functionalized; Membranes; Enhance; Antibody

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.

化学部的化学测量和成像项目获得了该奖项，该项目正在支持密歇根州立大学的Merlin Bruening和Xuefei Huang教授创建分离特定抗体并确定其序列的新方法。 抗体由于其特异性而作为治疗剂是有吸引力的，但是尽管其日益增长的医学重要性，新抗体的开发和表征是艰巨且昂贵的。 这项工作的目的是通过从混合物中捕获最强结合的天然抗体，然后对这些抗体进行测序，以使它们的合成能够用于未来的研究或应用，从而简化抗体的开发。 这些研究还将开发用于质量控制的抗体修饰鉴定方法。 该项目将涉及高中，本科和研究生。 此外，计划将研究概念，先进的质谱和表面分析技术纳入本科生，特别是研究生课程。 一个针对高中教师的研讨会将重点关注MS的蛋白质分析，以进一步扩大这一努力的影响，而少数民族本科生和高中生的实习应该有助于增加进入研究管道的代表性不足的学生的数量。这个项目探索膜-基于抗体的消化来控制蛋白水解肽的大小，并使用质谱法（MS）增强抗体的表征和测序。 蛋白酶如胃蛋白酶在膜孔中的固定化产生酶反应器，并且因为含蛋白酶的膜仅100微米厚，通过膜的高流速产生毫秒消化时间。 该研究考察了将来自msec digestion的大肽和来自较长digestion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蛋白质测序的肽排列。 与其他消化方法不同，基于膜的消化用相同的酶产生长肽和短肽，因此短肽的分子质量相加得到长肽的质量。 这将导致一种独特而有效的方法，用于基于这些大肽和小肽的质量之间的关系来排列蛋白质序列中的肽。 预期随后的大肽和小肽的MS/MS分析给出抗体序列的几乎完全的氨基酸覆盖。 此外，来自用胃蛋白酶修饰的膜的廉价消化的大肽将促进抗体氧化、糖基化和脱酰胺化的表征，甚至在混合物中。重要的是，在含胃蛋白酶的膜中消化是廉价的，并且在低pH下发生以最小化抗体降解。这些研究还将检查含有抗原的多孔膜是否可以从多克隆混合物中分离出一些紧密结合或快速结合的抗体。 与基于珠粒的分离方法相比，膜提供更低的死体积、更少的非特异性吸附以及对结合时间和冲洗的更精细控制，以潜在地促进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合成和应用成为可能。 因此，这项研究中探索的技术可能最终导致从免疫或感染的动物中发现和测序抗体的方法。