Skip to main content
Figure 2 | Cell Communication and Signaling

Figure 2

From: Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

Figure 2

MS/MS spectra of ADH1 containing non-oxidised (A) and oxidised (B) methionine residues. Three biological replicates of 10 μM 8-Br-cGMP-treated cells and H2O mock treated controls were collected at 0, 30 and 60 min. Proteins were precipitated using 10% (w/v) trichloroacetic acid in acetone, re-solubilised in 7 M urea, 2 M thiourea and 4% (w/v) CHAPS, reduced, alkylated and trypsin digested. Peptides were fractionated by cation exchange chromatography. Methionine oxidised peptides were enriched using TiO2 beads and re-suspended in 5% (v/v) acetonitrile and 0.1% (v/v) formic acid prior to identification and quantitation by LTQ Orbitrap coupled with a nanoelectrospray ion source. Peptides (5 μL) were injected onto a 50 mm × 0.3 mm Magic C18AQ column. The top 10 precursor ions were selected with a resolution of 60,000 for fragmentation using normalized collision-induced dissociation set at 35.0. Spectra were searched against TAIR using MASCOT, with a precursor mass tolerance of 10 ppm, a fragment ion mass tolerance of 0.3 Da, one missed cleavage, carbamidomethyl cysteine residues as fixed modification and oxidation and dioxidation of methionine residues as variable modifications. Proteins with a score > 95% were considered positively identified (corresponding score ≤ 31). Spectra were further processed with the Scaffold software using the “Trans-Proteomic Pipeline” algorithm (threshold 95%). Oxidised Met residues showed an increase in mass/charge ratio (m/z) of 15.9994. Arrows show Met residues at position 13 in the fragment DHDKPIQQVIAEMTDGGVDR of AT1G77120 before oxidation (m/z ratio 850.3723) (A) and after oxidation (m/z ratio 866.3673) (B).

Back to article page