Biochemistry and Physiology of Plants, Faculty of Biology, Bielefeld University, Bielefeld, Germany.
Methods Mol Biol. 2024;2832:99-113. doi: 10.1007/978-1-0716-3973-3_7.
Redox modulation is a common posttranslational modification to regulate protein activity. The targets of oxidizing agents are cysteine residues (Cys), which have to be exposed at the surface of the proteins and are characterized by an environment that favors redox modulation. This includes their protonation state and the neighboring amino acids. The Cys redox state can be assessed experimentally by redox titrations to determine the midpoint redox potential in the protein. Exposed cysteine residues and putative intramolecular disulfide bonds can be predicted by alignments with structural data using dedicated software tools and information on conserved cysteine residues. Labeling with light and heavy reagents, such as N-ethylmaleimide (NEM), followed by mass spectrometric analysis, allows for the experimental determination of redox-responsive cysteine residues. This type of thiol redox proteomics is a powerful approach to assessing the redox state of the cell, e.g., in dependence on environmental conditions and, in particular, under abiotic stress.
氧化还原调节是一种常见的翻译后修饰，用于调节蛋白质活性。氧化剂的靶标是半胱氨酸残基（Cys），这些残基必须暴露在蛋白质的表面，其环境有利于氧化还原调节。这包括它们的质子化状态和相邻的氨基酸。可以通过氧化还原滴定实验来评估 Cys 氧化还原状态，以确定蛋白质的中点氧化还原电位。可以使用专用软件工具和关于保守半胱氨酸残基的结构数据对齐来预测暴露的半胱氨酸残基和可能的分子内二硫键。用轻试剂和重试剂（如 N-乙基马来酰亚胺（NEM））标记，然后进行质谱分析，可用于实验测定氧化还原反应性半胱氨酸残基。这种类型的硫醇氧化还原蛋白质组学是评估细胞氧化还原状态的一种有力方法，例如，依赖于环境条件，特别是非生物胁迫。
