Martelet Armelle, Garrigue Valerie, Le Borgne Hélène, Borel Claire, Alexandre Sylvie, Crépin Ronan, Genet Bruno, Liu Haichuan, Zhang Yuzhuo, Clavier Séverine
Sanofi R&D, Vitry-sur-Seine 94400, France.
SCIEX, 1201 Radio Road, Redwood City, California 94065, United States.
J Am Soc Mass Spectrom. 2025 May 7;36(5):952-960. doi: 10.1021/jasms.4c00432. Epub 2025 Mar 25.
Mass spectrometry (MS) is a tool of choice for the in-depth characterization of new biotherapeutic molecules such as a complex naturally derived trispecific antibody (tsAb) that presents a tyrosine sulfation within the variable domain. Although tyrosine sulfation is an important post-translational modification responsible for strengthening protein-protein interactions, its localization is challenging, as the sulfate group is very labile using conventional positive ion mode fragmentation techniques. In this work, we describe the combination of functional testing and MS-based methods to study the impact of tyrosine sulfation in the tsAb. The presence of sulfation was confirmed by intact mass and peptide mapping analyses. For unambiguous localization of the sulfate group, electron activated dissociation (EAD) MS/MS experiments were employed to generate diagnostic fragments carrying an intact sulfate group. We also demonstrated that a significant decrease in binding of the tsAb to the target antigen was observed following the sulfatase treatment. Taken together, the results from this study support the notion that tyrosine sulfation plays an important role in protein-protein interactions.
质谱（MS）是深入表征新型生物治疗分子的首选工具，例如一种复杂的天然来源三特异性抗体（tsAb），其可变区内存在酪氨酸硫酸化修饰。尽管酪氨酸硫酸化是一种重要的翻译后修饰，有助于加强蛋白质-蛋白质相互作用，但其定位具有挑战性，因为使用传统的正离子模式裂解技术时，硫酸根基团非常不稳定。在这项工作中，我们描述了功能测试和基于质谱的方法相结合，以研究酪氨酸硫酸化对三特异性抗体的影响。通过完整质量分析和肽图谱分析证实了硫酸化的存在。为了明确硫酸根基团的定位，采用电子活化解离（EAD）串联质谱实验来生成携带完整硫酸根基团的诊断性片段。我们还证明，在硫酸酯酶处理后，观察到三特异性抗体与靶抗原的结合显著降低。综上所述，本研究结果支持酪氨酸硫酸化在蛋白质-蛋白质相互作用中起重要作用这一观点。
