Utilization of high-resolution mass spectrometry and data-independent analysis to track the monoclonal antibody spatial stability

Chemical cross-linking coupled with mass spectrometry (CXMS) offers the distance constraints critical for building the structural model of protein and protein complexes and understanding dynamics of biological systems. Originally developed for protein structural models, CXMS has evolved into method for studying protein complex formation, ligand-induced conformational changes, and quantitative structural analysis using isotopically labeled cross-linkers.
In this study we tested the potential of isotopically labeled MS-cleavable cross-linker to track the stability of the therapeutic monoclonal antibodies.
A novel isotopically labeled MS-cleavable cross-linker was synthesized, and its reactivity was successfully tested on peptide and protein standards. Further, the novel cross-linker was utilized to test the stability of selected therapeutical monoclonal antibodies, bevacizumab and trastuzumab, adopting the data-independent acquisition.
This study reports the advantages of using combination of 13C isotopically labeled MS-cleavable cross-linkers and data-independent mass spectrometry analysis for the automated identification of cross-linked products and thus monitoring the structural rearrangement of protein structure.

化学交联结合质谱技术（Chemical cross-linking coupled with mass spectrometry，CXMS）可为蛋白质及蛋白质复合物的结构模型构建、生物系统动力学解析提供关键的距离约束信息。该技术最初专为蛋白质结构模型开发，如今已发展为可用于研究蛋白质复合物形成、配体诱导构象变化，以及采用同位素标记交联剂开展定量结构分析的技术手段。
本研究针对同位素标记的质谱可裂解交联剂的潜力展开测试，以追踪治疗性单克隆抗体的稳定性。本研究合成了一种新型同位素标记的质谱可裂解交联剂，并在肽段与蛋白质标准品上成功验证了其反应活性。进一步地，本研究采用数据非依赖性采集（data-independent acquisition）策略，利用该新型交联剂对选定的治疗性单克隆抗体贝伐珠单抗（bevacizumab）和曲妥珠单抗（trastuzumab）的稳定性进行了测试。本研究证实，将13C同位素标记的质谱可裂解交联剂与数据非依赖性质谱分析相结合，可实现交联产物的自动化鉴定，进而实现蛋白质结构重排的动态监测。