Although incredibly diverse in specificity, millions of unique Immunoglobulin G (IgG) molecules in the human antibody repertoire share most of their amino acid sequence. These constant parts of IgG do not yield any useful information in attempts to sequence antibodies de novo. Therefore, methods focusing solely on the variable regions and providing unambiguous sequence reads are strongly advantageous. We report a mass spectrometry-based method that uses electron capture dissociation (ECD) to provide straightforward-to-read sequence ladders for the variable parts of both the light and heavy chains, with a preference for the functionally important CDR3. We optimized this method on the therapeutic antibody Trastuzumab and demonstrate its applicability on two monoclonal quartets of the four IgG subclasses, IgG1, IgG2, IgG3 and IgG4. The method is based on proteolytically separating the variable F(ab′)₂ part from the conserved Fc part, whereafter the F(ab′)₂ portions are mass-analyzed and fragmented by ECD. Pure ECD, without additional collisional activation, leads to straightforward-to-read sequence tags covering the CDR3 of both the light and heavy chains. Using molecular modelling and structural analysis, we discuss and explain this selective fragmentation behavior and describe how structural features of the different IgG subclasses lead to distinct fragmentation patterns. Overall, we foresee that pure ECD on F(ab′)₂ or Fab molecules can become a valuable tool for the de novo sequencing of serum antibodies.

中文翻译：

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从头进行IgG测序的覆盖范围选择性

尽管特异性异常多种多样，但人类抗体库中数百万个独特的免疫球蛋白G（IgG）分子共享其大部分氨基酸序列。IgG的这些恒定部分在尝试从头测序抗体时不会产生任何有用的信息。因此，仅关注可变区并提供明确的序列读数的方法是非常有利的。我们报告了一种基于质谱的方法，该方法使用电子捕获解离（ECD）为轻链和重链的可变部分提供直观易懂的序列阶梯，并优先选择功能上重要的CDR3。我们在治疗性抗体曲妥珠单抗上优化了该方法，并证明了其在四个IgG亚类IgG1，IgG2，IgG3和IgG4的两个单克隆四元组中的适用性。该方法基于将蛋白的F（ab'）₂部分与保守的Fc部分蛋白水解分离，此后F（ab'）₂部分通过ECD进行质量分析和分段。纯ECD，无需额外的碰撞激活，可导致直接读取的序列标签覆盖轻链和重链的CDR3。使用分子建模和结构分析，我们讨论并解释了这种选择性片段化行为，并描述了不同IgG亚类的结构特征如何导致不同的片段化模式。总体而言，我们预见到，F（ab'）₂或Fab分子上的纯ECD可能成为从头测序血清抗体的有价值的工具。