利用高分辨率质谱和数据独立分析来跟踪单克隆抗体的空间稳定性。

IF 3.8 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Expert Review of Proteomics Pub Date : 2025-04-20 DOI:10.1080/14789450.2025.2492763

Valerie Prochazkova, Michal Rosulek, Michael Karpisek, Vaclav Matousek, Petr Novak, Zdenek Kukacka

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引用次数: 0

摘要

目的：化学交联联用质谱（CXMS）为建立蛋白质和蛋白质复合物的结构模型和理解生物系统的动力学提供了至关重要的距离限制。CXMS最初用于蛋白质结构模型，现已发展成为研究蛋白质复合物形成、配体诱导构象变化以及使用同位素标记交联剂进行定量结构分析的方法。方法：在本研究中，我们测试了同位素标记的ms可切割交联剂在追踪治疗性单克隆抗体稳定性方面的潜力。结果：合成了一种新型的同位素标记的ms可切割交联剂，并成功地在肽和蛋白质标准上进行了反应性测试。此外，采用数据独立获取的方法，利用新型交联剂测试了选定的治疗性单克隆抗体贝伐珠单抗和曲妥珠单抗的稳定性。结论：本研究报道了将13C同位素标记的ms可切割交联剂与数据独立质谱分析相结合用于交联产物的自动鉴定，从而监测蛋白质结构的结构重排的优势。

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

Objectives: 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.

Methods: In this study we tested the potential of isotopically labeled MS-cleavable cross-linker to track the stability of the therapeutic monoclonal antibodies.

Results: 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.

Conclusion: 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.

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来源期刊

Expert Review of Proteomics 生物-生化研究方法

CiteScore

7.60

自引率

0.00%

发文量

审稿时长

6-12 weeks

期刊介绍： Expert Review of Proteomics (ISSN 1478-9450) seeks to collect together technologies, methods and discoveries from the field of proteomics to advance scientific understanding of the many varied roles protein expression plays in human health and disease. The journal coverage includes, but is not limited to, overviews of specific technological advances in the development of protein arrays, interaction maps, data archives and biological assays, performance of new technologies and prospects for future drug discovery. The journal adopts the unique Expert Review article format, offering a complete overview of current thinking in a key technology area, research or clinical practice, augmented by the following sections: Expert Opinion - a personal view on the most effective or promising strategies and a clear perspective of future prospects within a realistic timescale Article highlights - an executive summary cutting to the author''s most critical points.