Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, 1251 Wescoe Hall Dr, Lawrence, KS, 66045, USA.
Anal Bioanal Chem. 2018 Apr;410(10):2467-2484. doi: 10.1007/s00216-017-0772-1. Epub 2017 Dec 18.
Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure 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 bond 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 of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) 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 MS-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 bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-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 characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass spectrometry data, and software tools.
它们确保正确折叠、提供稳定性并确保正确的功能。随着蛋白质，特别是高度二硫键结合的单克隆抗体，在生物治疗中的应用越来越多，现在重要的是要确认正确的二硫键连接，并验证蛋白质治疗剂中二硫键变体的存在或不存在。这些研究有助于确保安全性和疗效。因此，二硫键是生物治疗药物开发过程中必须密切监测的蛋白质关键质量属性之一。然而，由于生物分子的复杂性，二硫键分析具有挑战性。质谱 (MS) 一直是用于此类复杂生物分子表征的首选分析工具，已经报道了几种方法来满足在蛋白质中二硫键作图的挑战性任务。在这篇综述中，我们描述了相关的、最近的基于 MS 的技术，并提供了在蛋白质中二硫键分析中有效进行的重要考虑因素。综述重点介绍了用于正确样品制备的方法、用于二硫键分析的片段化技术、基于这些片段化技术的最新二硫键作图方法，以及为从液相色谱-MS/MS 数据中快速分析二硫键而设计的自动化算法。从事蛋白质特性方法开发的研究人员可以使用本文中的信息来促进基于 MS 的蛋白质中二硫键分析新方法的开发。此外，特别是通过抗体中二硫键作图来表征生物治疗剂的人员，可以使用这篇综述来选择其生物制品中二硫键分配的最佳策略。
