Abstract
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 Arabidopsis thaliana 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.
Highlights
Despite their potentially harmful reactivity toward most biomolecules, reactive oxygen species (ROS) play key signaling roles in many biological processes in a wide range of organisms [1]
Consistent with a role in aging [30,41], several recent studies have pointed out the role of protein oxidation in seed longevity, notably including oxidation at Met residues in proteins [42,43,44]. To further document this question and toward a global characterization of oxidation of Met residues in proteins to methionine sulfoxide (MetO), we have developed a handy proteomic method called 2Dd-CNBr based on the use of two-dimensional diagonal electrophoresis (2Dd) and cyanogen bromide (CNBr) cleavage after the first dimension [45], followed by mass spectrometry for the identification of proteins of interest
We have developed a novel method coupling 2Dd using CNBr cleavage after the first protein electrophoresis and mass spectrometry to allow evidencing the oxidation of Met residues in proteins, at a high throughput, and on complex mixtures of proteins
Summary
Despite their potentially harmful reactivity toward most biomolecules, reactive oxygen species (ROS) play key signaling roles in many biological processes in a wide range of organisms [1]. Generated ROS, produced by electron transport chains, react with different macromolecules in the cell and cause oxidative damage. Such oxidative damage accumulates over time during the life cycle of many organisms, and has been suggested to be one possible cause of aging [2]. Protein oxidation is assumed to be an important process affecting seed germination and vigor [7,8]. The role of ROS in seed science has emerged in the last decade and it is well established that a regulated accumulation of ROS, which is referred to as the oxidative window for germination [11,12], is a prerequisite for radicle elongation, the first visible symptom of seed germination [11]
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