Quantitatively Differentiating Antibodies Using Charge-State Manipulation, Collisional Activation, and Ion Mobility-Mass Spectrometry
Quantitatively Differentiating Antibodies Using Charge-State Manipulation, Collisional Activation, and Ion Mobility-Mass Spectrometry
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DOI：
10.1021/acs.analchem.3c04638
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发表时间：
2023-12-26
影响因子：
7.4
通讯作者：
Bush，Matthew F.
中科院分区：
文献类型：
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作者：
Gozzo，Theresa A.;Bush，Matthew F.
基于抗体的治疗方法在产品数量和患者使用方面都在继续扩大。这些异质蛋白质挑战了传统的药物表征策略，但离子迁移率（IM）和质谱（MS）方法缓解了高阶结构表征的挑战。能量依赖的IM-MS，例如，碰撞诱导的去折叠（CIU）已被证明对结构的细微差异敏感。在这项研究中，我们结合联合收割机的电荷还原方法，阳离子到阴离子质子转移反应（CAPTR），与能量依赖的IM-MS和不同的溶液条件，以探讨其联合作用对人骨髓瘤IgG 1 κ和IgG 4 κ的气相结构。CAPTR与仅MS分析配对提高了电荷状态分配的置信度和干扰蛋白质种类的分辨率。碰撞截面分布，确定每个电荷减少的产品。相似性评分用于定量比较由分析两种抗体样品的匹配实验确定的分布。相对于使用能量依赖性IM-MS而不进行电荷状态操纵的工作流程，CAPTR和能量依赖性IM-MS的组合增强了这些抗体的分化。结合起来，这些结果表明CAPTR可以有益于抗体表征和分化的许多方面。
Antibody-based therapeutics continue to expand both in the number of products and in their use in patients. These heterogeneous proteins challenge traditional drug characterization strategies, but ion mobility (IM) and mass spectrometry (MS) approaches have eased the challenge of higher-order structural characterization. Energy-dependent IM-MS, e.g., collision-induced unfolding (CIU), has been demonstrated to be sensitive to subtle differences in structure. In this study, we combine a charge-reduction method, cation-to-anion proton-transfer reactions (CAPTR), with energy-dependent IM-MS and varied solution conditions to probe their combined effects on the gas-phase structures of IgG1κ and IgG4κ from human myeloma. CAPTR paired with MS-only analysis improves the confidence of charge-state assignments and the resolution of the interfering protein species. Collision cross-section distributions were determined for each of the charge-reduced products. Similarity scoring was used to quantitatively compare distributions determined from matched experiments analyzing samples of the two antibodies. Relative to workflows using energy-dependent IM-MS without charge-state manipulation, combining CAPTR and energy-dependent IM-MS enhanced the differentiation of these antibodies. Combined, these results indicate that CAPTR can benefit many aspects of antibody characterization and differentiation.