Epitope mapping by H/D exchange mass spectrometry

通过 H/D 交换质谱法进行表位作图

• 批准号: 7405730
• 负责人: Yoshitomo Hamuro
• 金额: $ 10万
• 依托单位: EXSAR CORPORATION
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2008-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7405730
• 关键词: Amides; Antibodies; Antibody Binding Sites; Antigen-Antibody Complex; Antigens; Automation; Back; Basic Science; Binding; Biological Assay; Biological Products; Biotechnology; Buffers; Businesses; Condition; Coupled; Crystallography; Deuterium; Digestion; End Point; Environment; Epitope Mapping; Epitopes; Equipment; Excision; Exclusion; G-substrate; Hydrogen; Immobilization; Incubated; Investigation; Knowledge; Laboratories; Ligands; Liquid substance; Maps; Mass Spectrum Analysis; Measurement; Measures; Methodology; Monitor; Numbers; Pattern; Peptide Fragments; Peptides; Pharmacologic Substance; Phase; Plant Resins; Production; Protein Dynamics; Proteins; Proteolysis; Public Health; Purpose; Reaction; Research; Resolution; Resources; Roentgen Rays; Services; Signal Transduction; Site-Directed Mutagenesis; Solutions; Solvents; Standards of Weights and Measures; Structure; System; Techniques; Technology; Temperature; Therapeutic Monoclonal Antibodies; Therapeutic antibodies; Time; Treatment Efficacy; X-Ray Crystallography; antigen binding; antigen processing; concept; cross reactivity; cytochrome c; day; instrument; instrumentation; miniaturize; mutant; pressure; prevent; protein protein interaction; research study; size

DESCRIPTION (provided by applicant): The aim of this research is to use hydrogen/deuterium (H/D) exchange coupled with proteolysis and mass spectrometry to map antigen-antibody interactions. While the epitope mapping of therapeutic monoclonal antibody is a critical step for the characterization of the antibody, approaches used to characterize antigen-antibody interactions, such as X-ray crystallography and site directed mutagenesis are target limited and labor intensive; not all proteins can be crystallized and site-directed mutagenesis requires the production of a significant number of mutants. Furthermore, other techniques, such as binding assay of overlapping peptides, may miss conformational epitopes. H/D exchange is not subject to these limitations. We propose to measure the H/D exchange of an antigen in the presence and absence of its antibody and compare the deuteration levels of each peptide fragment generated under these two conditions. The binding of an antibody to its antigen should retard the exchange of amide hydrogen through solvent exclusion, and/or restriction of conformational fluctuations at the antigen-antibody interface. The comparison of the antigen H/D exchange patterns in the presence and absence of its antibody should generate a "footprint" of the antibody binding site - the antigen epitope. H/D exchange is less disruptive and less resource intensive than traditional epitope mapping technologies. During exchange, the analyte protein retains its native solution state. On halting the reaction, the partially deuterated protein is then fragmented proteolytically to analyze deuterium incorporation of each peptide fragment by mass spectrometry. ExSAR has recently developed fully automated instrumentation to measure H/D exchange for protein dynamics and protein-ligand interactions. ExSAR would like to expand this capability to study protein- protein interactions and more specifically, antigen-antibody interactions. Our strategy requires antibody immobilization, creation of an antibody column, and execution of the antigen H/D exchange in the antibody column. The confirmation of the crystallographic contact residues by the H/D exchange determined epitope will validate this strategy. Our long-term objective is to develop a fully automated instrument capable of measuring H/D exchange of an antigen in the presence or absence of antibody. PUBLIC HEALTH RELEVANCE - PROJECT NARRATIVE: The epitope mapping of therapeutic monoclonal antibody is a critical step for the characterization of the antibody. ExSAR's developing technologies could be applicable to the investigation of therapeutic antibodies. A detailed knowledge of the dynamic interaction of an antibody and its antigen may contribute towards an understanding of mechanisms underlying antibody cross-reactivity, an important factor in therapeutic efficacy. Establishing a fast, cheap and reliable methodology for the epitope mapping should have a direct impact on the monoclonal antibody therapeutic business.

描述（由申请人提供）：本研究的目的是使用氢/氘（H/D）交换结合蛋白水解和质谱法来绘制抗原-抗体相互作用。虽然治疗性单克隆抗体的表位作图是表征抗体的关键步骤，但是用于表征抗原-抗体相互作用的方法，例如X射线晶体学和定点诱变是目标有限的和劳动密集型的;不是所有蛋白质都可以结晶，并且定点诱变需要产生大量突变体。此外，其他技术，如重叠肽的结合测定，可能会错过构象表位。H/D交换不受这些限制。我们建议在存在和不存在其抗体的情况下测量抗原的H/D交换，并比较在这两种条件下产生的每个肽片段的氘化水平。抗体与其抗原的结合应通过溶剂排斥和/或限制抗原-抗体界面处的构象波动来延迟酰胺氢的交换。在存在和不存在其抗体的情况下抗原H/D交换模式的比较应产生抗体结合位点-抗原表位的“足迹”。H/D交换比传统的表位作图技术具有更小的破坏性和更少的资源密集性。在交换过程中，分析物蛋白质保持其天然溶液状态。在停止反应时，然后将部分氘代的蛋白质蛋白水解片段化，以通过质谱法分析每个肽片段的氘掺入。ExSAR最近开发了全自动仪器来测量蛋白质动力学和蛋白质-配体相互作用的H/D交换。ExSAR希望扩大这种能力，以研究蛋白质-蛋白质相互作用，更具体地说，抗原-抗体相互作用。我们的策略需要抗体固定化，抗体柱的创建，以及在抗体柱中进行抗原H/D交换。通过H/D交换确定的表位确认晶体学接触残基将验证该策略。我们的长期目标是开发一种能够在存在或不存在抗体的情况下测量抗原的H/D交换的全自动仪器。 公共卫生相关性-项目叙述：治疗性单克隆抗体的表位作图是抗体表征的关键步骤。ExSAR开发的技术可适用于治疗性抗体的研究。抗体及其抗原的动态相互作用的详细知识可能有助于理解抗体交叉反应性的潜在机制，这是治疗效果的重要因素。建立一种快速、廉价和可靠的表位定位方法将对单克隆抗体治疗业务产生直接影响。