Boyatzis Amber E, Bringans Scott D, Piggott Matthew J, Duong Marisa N, Lipscombe Richard J, Arthur Peter G
School of Chemistry and Biochemistry, University of Western Australia , Crawley, Western Australia 6009, Australia.
Proteomics International , Perth, Western Australia 6009, Australia.
J Proteome Res. 2017 May 5;16(5):2004-2015. doi: 10.1021/acs.jproteome.6b01060. Epub 2017 Apr 7.
Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (∼14%) of gels and proteolytic digestion buffers (∼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.
由活性氧和氮物种（RONS）引起的氧化应激在许多疾病的病理生理学中起着重要作用。RONS的一个关键靶点是蛋白质半胱氨酸残基的硫醇基团。由于硫醇氧化会影响蛋白质功能，因此通过鉴定氧化蛋白质可以确定氧化应激如何影响组织功能的机制信息。所使用的探针必须具有特异性和敏感性，例如用于还原型半胱氨酸硫醇烷基化的马来酰亚胺。然而，我们发现马来酰亚胺烷基化肽（MAPs）在蛋白质组学研究常见的样品制备条件下会被氧化和水解。这可能导致高达90%的MAP信号转化为氧化或水解的MAPs，从而降低分析的灵敏度。这些修饰的很大一部分可通过考马斯亮蓝银染色（约14%）的凝胶和蛋白水解消化缓冲液（约20%）来解释。在凝胶电泳过程中使用巯基乙酸、凝胶中的三氯乙醇-紫外线蛋白质可视化以及pH值为7.0的TRIS蛋白水解消化缓冲液，可保留超过40%的MAP信号。这项工作表明，通过改变样品制备流程可以减少对MAPs的修饰，增强马来酰亚胺在鉴定氧化肽方面的潜在实用性。
