Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here we demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by LC-MS/MS in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron transfer high-energy collision dissociation (EThcD) on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody Herceptin, with an accuracy of 99% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.

中文翻译：

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基于质谱的抗FLAG-M2抗体测序，使用多种蛋白酶和双重片段化方案

抗体序列信息对于理解抗原结合的结构基础至关重要，并能将抗体用作治疗和研究工具。在这里，我们展示了一种直接从纯化抗体产物中进行单克隆IgG的从头测序的方法。该方法使用一组多种互补蛋白酶来产生合适的肽，以自下而上的方式通过LC-MS / MS进行从头测序。此外，我们在所有肽前体上均采用了阶梯式高能碰撞解离（阶梯式HCD）和电子转移高能碰撞离解（EThcD）的双重裂解方案。该方法实现了单克隆抗体赫赛汀的全序列覆盖，在可变区的准确性为99％。我们应用该方法对广泛使用的抗FLAG-M2小鼠单克隆抗体进行测序，我们通过重塑Fab的高分辨率晶体结构并在Western blot分析中证明与FLAG标记的靶蛋白的结合成功地验证了该方法。该方法因此提供了单克隆抗体的鲁棒且可靠的序列。