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使用氢氘交换质谱法测定曲妥珠单抗和帕妥珠单抗与选择性亲和配体的结合位点。

Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry.

机构信息

Analytical Biochemistry, Department of Pharmacy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Novartis Technical Research & Development Biologics, Hexal AG, Keltenring 1 + 3, 82041 Oberhaching, Germany.

出版信息

J Am Soc Mass Spectrom. 2023 Apr 5;34(4):775-783. doi: 10.1021/jasms.3c00069. Epub 2023 Mar 24.

DOI:10.1021/jasms.3c00069
PMID:36960982
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10080681/
Abstract

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding. Bottom-up HDX-MS experiments including online pepsin digestion revealed that the binding sites of the affimer reagents were mainly located in the complementarity-determining region (CDR) 2 of the heavy chain of the respective antibodies. Three-dimensional models of the binding interaction between the affimer reagents and the antibodies were built by homology modeling and molecular docking based on the HDX data.

摘要

氢氘交换质谱（HDX-MS）是一种探测蛋白质或蛋白配体复合物中可交换酰胺氢原子的溶剂可及性和构象动力学的方法。本文介绍了 HDX-MS 在确定亲和素试剂与曲妥珠单抗和帕妥珠单抗单克隆抗体结合位点中的应用。抗体-亲和素复合物的完整和亚基水平的 HDX-MS 分析表明，亲和素结合后抗体 Fab 区域的 HDX 显著受到保护。基于 HDX 数据的自上而下的 HDX-MS 实验，包括在线胃蛋白酶消化，揭示了亲和素试剂的结合位点主要位于相应抗体重链的互补决定区（CDR）2。基于 HDX 数据，通过同源建模和分子对接构建了亲和素试剂与抗体之间结合相互作用的三维模型。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb8d/10080681/4e436c969e7d/js3c00069_0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb8d/10080681/7d8c7a542164/js3c00069_0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb8d/10080681/86ae2783c030/js3c00069_0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb8d/10080681/19f1b1623afe/js3c00069_0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb8d/10080681/4e436c969e7d/js3c00069_0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb8d/10080681/7d8c7a542164/js3c00069_0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb8d/10080681/86ae2783c030/js3c00069_0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb8d/10080681/19f1b1623afe/js3c00069_0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb8d/10080681/4e436c969e7d/js3c00069_0004.jpg

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