Epitope mapping by H/D exchange mass spectrometry

描述（由申请人提供）：本研究的目的是利用氢/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定位是抗体表征的关键步骤，但用于表征抗原-抗体相互作用的方法，如x射线晶体学和定点诱变，目标有限且劳动密集型；并不是所有的蛋白质都可以结晶，定点诱变需要产生大量的突变体。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兑换不受这些限制。我们建议测量抗原在存在和不存在抗体时的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-抗体界面的构象波动来延缓酰胺氢的交换。在存在和不存在其抗体的情况下，抗原H/D交换模式的比较应该产生抗体结合位点的“足迹”-抗原表位。与传统的表位定位技术相比，H/D交换的破坏性较小，资源密集程度也较低。在交换过程中，被分析蛋白保持其天然溶液状态。停止反应后，将部分氘化的蛋白质进行蛋白水解，通过质谱分析每个肽片段的氘结合情况。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希望将这种能力扩展到研究蛋白质-蛋白质相互作用，更具体地说，抗原-抗体相互作用。我们的策略需要固定抗体，创建抗体柱，并在抗体柱中执行抗原H/D交换。通过H/D交换确定的表位对晶体接触残基的确认将验证这一策略。我们的长期目标是开发一种全自动仪器，能够在存在或不存在抗体的情况下测量抗原的H/D交换。

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.