Institute of Life Technologies, University of Applied Sciences Western Switzerland (HES-SO Valais/Wallis), 1950 Sion, Switzerland.
Expert Rev Proteomics. 2013 Oct;10(5):489-501. doi: 10.1586/14789450.2013.837663. Epub 2013 Oct 3.
Elucidating disulfide linkage patterns is a crucial part of protein characterization, for which mass spectrometry (MS) is now an indispensable analytical tool. In many cases, MS-based disulfide connectivity assignment is straightforwardly achieved using one-step protein fragmentation in the unreduced form followed by mass measurement of bridged fragments. By contrast, venom proteins, which are receiving increasing interest as potential therapeutics, are a challenge for MS-based disulfide assignment due to their numerous closely spaced cysteines and knotted disulfide structure, requiring creative strategies to determine their connectivity. Today, these include the use of an array of reagents for enzymatic and/or chemical cleavage, partial reduction, differential cysteine labeling and tandem MS. This review aims to describe the toolkit of techniques available to MS users approaching both straightforward and complex disulfide bridge assignments, with a particular focus on strategies utilizing standard instrumentation found in a well-equipped analytical or proteomics laboratory.
阐明二硫键连接模式是蛋白质表征的关键部分，为此，质谱 (MS) 现在是一种不可或缺的分析工具。在许多情况下，使用一步法在未还原形式下对蛋白质进行片段化，然后测量桥接片段的质量，就可以直接基于 MS 进行二硫键连接性分配。相比之下，毒液蛋白作为潜在的治疗药物越来越受到关注，但由于其具有大量紧密间隔的半胱氨酸和打结的二硫键结构，因此对基于 MS 的二硫键分配构成了挑战，需要创造性的策略来确定其连接性。如今，这些策略包括使用一系列用于酶切和/或化学切割、部分还原、差异半胱氨酸标记和串联 MS 的试剂。本综述旨在描述可用于处理简单和复杂二硫键桥接分配的 MS 用户的技术工具包，特别关注利用配备齐全的分析或蛋白质组学实验室中标准仪器的策略。
