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Top-Down Characterization of an Intact Monoclonal Antibody Using Activated Ion Electron Transfer Dissociation.
Analytical Chemistry ( IF 7.4 ) Pub Date : 2020-07-01 , DOI: 10.1021/acs.analchem.0c00705
Jean M. Lodge , Kevin L. Schauer , Dain R. Brademan , Nicholas M. Riley , Evgenia Shishkova , Michael S. Westphall , Joshua J. Coon

Monoclonal antibodies (mAbs) are important therapeutic glycoproteins, but their large size and structural complexity make them difficult to rapidly characterize. Top-down mass spectrometry (MS) has the potential to overcome challenges of other common approaches by minimizing sample preparation and preserving endogenous modifications. However, comprehensive mAb characterization requires generation of many, well-resolved fragments and remains challenging. While ETD retains modifications and cleaves disulfide bonds—making it attractive for mAb characterization—it can be less effective for precursors having high m/z values. Activated ion electron transfer dissociation (AI-ETD) uses concurrent infrared photoactivation to promote product ion generation and has proven effective in increasing sequence coverage of intact proteins. Here, we present the first application of AI-ETD to mAb sequencing. For the standard NIST mAb, we observe a high degree of complementarity between fragments generated using standard ETD with a short reaction time and AI-ETD with a long reaction time. Most importantly, AI-ETD reveals disulfide-bound regions that have been intractable, thus far, for sequencing with top-down MS. We conclude AI-ETD has the potential to rapidly and comprehensively analyze intact mAbs.

中文翻译:

使用活化离子电子转移解离对完整单克隆抗体进行自上而下的表征。

单克隆抗体 (mAb) 是重要的治疗性糖蛋白,但它们的大尺寸和结构复杂性使其难以快速表征。自上而下的质谱 (MS) 有可能通过最小化样品制备和保留内源性修饰来克服其他常见方法的挑战。然而,全面的 mAb 表征需要生成许多分离良好的片段,并且仍然具有挑战性。虽然 ETD 保留修饰并切割二硫键——使其对 mAb 表征具有吸引力——但它对具有高m / z的前体可能不太有效价值观。活化离子电子转移解离 (AI-ETD) 使用并发红外光活化来促进产物离子的产生,并已证明可有效增加完整蛋白质的序列覆盖率。在这里,我们介绍了 AI-ETD 在 mAb 测序中的首次应用。对于标准 NIST mAb,我们观察到使用具有短反应时间的标准 ETD 和具有长反应时间的 AI-ETD 生成的片段之间的高度互补性。最重要的是,AI-ETD 揭示了迄今为止难以用自上而下 MS 测序的二硫键结合区域。我们得出结论,AI-ETD 具有快速全面分析完整 mAb 的潜力。
更新日期:2020-08-04
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