Kalemba Ewa Marzena, Valot Benoît, Job Dominique, Bailly Christophe, Meimoun Patrice
Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland.
UMR 7622 Biologie du Développement, IBPS, Sorbonne Université, CNRS, F-75005 Paris, France.
Plants (Basel). 2022 Feb 21;11(4):569. doi: 10.3390/plants11040569.
In recent years, several reports pointed out the role of protein oxidation in seed longevity, notably regarding the oxidation of methionine (Met) residues to methionine sulfoxide (MetO) in proteins. To further consider this question, we present a handy proteomic method based on the use of two-dimensional diagonal electrophoresis (2Dd) and cyanogen bromide (CNBr) cleavage, which we refer to as 2Dd-CNBr. CNBr treatment of proteins causes the non-enzymatic hydrolysis of peptide bonds on the carboxyl side of reduced Met residues. However, Met oxidation causes a lack of cleavage, thus modifying the electrophoretic mobility of CNBr-induced peptides. This approach was first validated using bovine serum albumin as a model protein, which confirmed the possibility of distinguishing between oxidized and non-oxidized forms of Met-containing peptides in gels. Then, the 2Dd-CNBr method was applied to the seed protein extract in a control (non-oxidized) condition and in an oxidized one (as obtained following hypochlorous acid treatment). Twenty-four oxidized Met residues in 19 proteins identified by mass spectrometry were found to be surface exposed in these proteins. In the three-dimensional environment of the oxidized Met, we detected amino acid residues that could be converted by oxidation (carbonylation) or by phosphorylation, suggesting a possible interplay between Met oxidation and the other protein modifications. The identification of the proteins oxidatively modified in Met residues revealed the finding that MetO-containing proteins are related to seed longevity. Based on these results, we suggest that the method presently described also has the potential for wider applications.
近年来，多篇报道指出蛋白质氧化在种子寿命中的作用，特别是蛋白质中甲硫氨酸（Met）残基氧化为甲硫氨酸亚砜（MetO）方面。为了进一步探讨这个问题，我们提出了一种简便的蛋白质组学方法，该方法基于二维对角线电泳（2Dd）和溴化氰（CNBr）裂解，我们将其称为2Dd-CNBr。蛋白质经CNBr处理会导致还原型Met残基羧基侧肽键的非酶促水解。然而，Met氧化会导致裂解缺失，从而改变CNBr诱导肽段的电泳迁移率。该方法首先以牛血清白蛋白作为模型蛋白进行验证，证实了在凝胶中区分含Met肽段的氧化型和非氧化型的可能性。然后，将2Dd-CNBr方法应用于处于对照（未氧化）条件和氧化条件（次氯酸处理后获得）下的种子蛋白提取物。通过质谱鉴定出的19种蛋白质中的24个氧化型Met残基在这些蛋白质中被发现位于表面。在氧化型Met的三维环境中，我们检测到了可通过氧化（羰基化）或磷酸化进行转化的氨基酸残基，这表明Met氧化与其他蛋白质修饰之间可能存在相互作用。对Met残基发生氧化修饰的蛋白质的鉴定揭示了含MetO的蛋白质与种子寿命相关这一发现。基于这些结果，我们认为目前所描述的方法也具有更广泛的应用潜力。
