Are Methionine Sulfoxide-Containing Proteins Related to Seed Longevity? A Case Study of Arabidopsisthaliana Dry Mature Seeds Using Cyanogen Bromide Attack and Two-Dimensional-Diagonal Electrophoresis

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.

Keywords: mass spectrometry; methionine sulfoxide; oxidative stress; post-translational modifications; protein modification; redox proteomics; seed viability; two-dimensional diagonal electrophoresis.

Figure 1

Schematic representation of the 2Dd strategy using CNBr cleavage after the first electrophoretic dimension and presentation of the expected results. (1) SDS-PAGE of protein mixtures; (2) the gel strip is cut out and incubated with agitation for 30 min in CNBr cleavage buffer at room temperature (approx. 22 °C). The gel strip is rinsed briefly in deionized water and washed twice for 5 min in water. Then, the gel strip is equilibrated twice for 10 min in an equilibration solution containing dithiothreitol (DTT) and iodoacetamide (IAA); (3), the gel strip is placed on top of acrylamide gel to perform a second-dimensional separation by SDS-PAGE. (A) Control condition (reduced); (B) oxidizing condition; red dotted circles and red circles represent reduced and oxidized form of proteins, respectively.

Figure 2

2Dd-CNBr analysis of BSA. (A) Theoretical profile of fully non-oxidized BSA after 2Dd-CNBr; (B) experimental profile obtained with 100 ng BSA in non-oxidizing conditions (control); (C) experimental profile obtained with 100 ng BSA oxidized by HOCl in gel; (D) experimental profile obtained with 10 µg BSA in the non-oxidizing condition. Proteins were visualized by silver staining. Molecular mass markers are indicated for both dimensions.

Figure 3

1D and 2Dd-CNBr analyses of 40-µg soluble protein extracts from mature Arabidopsis seeds. (A) 1D separation of Arabidopsis seed proteins; (B) 2Dd-CNBr (control non-oxidized condition). Proteins sre visualized by silver staining; (C) as in (B), where red crosses indicate detected spots by using Image Master Platinum software (GE Healthcare, Chicago, IL, USA). Molecular mass markers are indicated for both dimensions.

Figure 4

2Dd-CNBr analysis of 40-µg soluble protein extracts from mature Arabidopsis seeds, in control (non-oxidized) and oxidized (HOCl) conditions. (A) 2Dd-CNBr (control non-oxidized condition); (B) 2Dd-CNBr (in gel oxidized proteins). Proteins are visualized by silver staining. Molecular mass markers are indicated for both dimensions. Red marks represent proteins analyzed by mass spectrometry.

Figure 5

Methionine content analysis of identified peptides by mass spectrometry after 1D SDS-PAGE (A), 2Dd-CNBr carried out in control (non-oxidized) (B) and oxidized (HOCl) (C) conditions. (A) 5968 peptides were identified by two independent mass spectrometry analyses; 1348 peptides contained at least one methionine including 757 and 1109 oxidized and reduced Met residues, respectively (see Table S6). (B) 704 peptides were identified and 29 contained at least an oxidized (+15.99491 Da) and 23 a modified Met residue (−29.9928 Da or −48.0034 Da) (see Table S3). (C) 378 peptides were identified and 110 contained at least one oxidized Met residue (+15.99491 Da) (see Table S5). HS, homoserine.

Figure 6

3D model of Arabidopsis CRA1 protein (AT5G44120). The model was built using Jmol software. The white arrow in the green dotted circles show the oxidized Met position; blue areas show solvent-accessible surface.