Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins.
Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins.
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DOI：
10.1007/s00216-017-0772-1
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发表时间：
2018-04
影响因子：
4.3
通讯作者：
Desaire H
中科院分区：
文献类型：
--
作者：
Lakbub JC;Shipman JT;Desaire H
二硫键是蛋白质的重要结构部分；它们确保适当的折叠，提供稳定性和适当的功能。随着生物治疗中越来越多地使用蛋白质，特别是单克隆抗体，它们是高度二硫键结合的，现在重要的是确认正确的二硫键连通性，并验证蛋白质治疗中是否存在二硫键变异体。这些研究有助于确保安全性和有效性。因此，在生物治疗药物的开发过程中，二硫键是蛋白质的关键质量属性（cqa）之一，必须密切监测。然而，由于生物分子的复杂性，二硫键分析具有挑战性。质谱分析一直是表征这类复杂生物分子的首选分析工具，据报道，有几种方法可以满足绘制蛋白质中二硫键的挑战性任务。在这篇综述中，我们描述了相关的，最近的基于质谱的技术，并提供了有效的蛋白质二硫键分析所需的重要考虑因素。综述的重点是合适的样品制备方法、用于二硫分析的碎片化技术、基于碎片化技术的二硫键映射方法，以及用于从LC-MS/MS数据中快速分析二硫键的自动算法。参与蛋白质表征方法开发的研究人员可以使用这里的信息来促进开发新的基于质谱的蛋白质二硫键分析方法。此外，从事生物疗法表征，特别是抗体中二硫键定位的个体，可以使用这篇综述来选择其生物制品二硫键分配的最佳策略。
Disulfide bonds are important structural moieties of proteins; they ensure proper folding, provide stability, and proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes (CQAs) of proteins that have to be monitored closely during the biotherapeutics’ development. However, disulfide bond analysis is challenging due to the complexity of the biomolecules. Mass spectrometry has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent mass spectrometry-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from LC-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals doing biotherapeutics characterization, especially disulfide bond mapping in antibodies, can use this review to choose best strategies for disulfide bond assignment of their biologic products.
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影响因子：
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作者：
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通讯作者：
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