Extended Data Fig. 4: H2O2 impact on RAP22-15 peptide oxidation via non-enzymatic N-terminal cysteine modification and AtPCO1/2 inhibition.
From: H2O2 repurposes plant O2 sensing to regulate post-hypoxia responses
(a) Quantification of oxidised RAP22-15 species after direct H2O2 treatment (1 mM) of 200 µM peptide in real time by RapidFire mass spectrometry reveals ~4.5 µM sulfenic (SOH), ~7 µM sulfinic (SO2H) and ~1 µM sulfonic acid (SO3H) on the N-terminal cysteine residue after 1 h compared to PCO-catalysed oxidation of RAP22-15 (>30 µM after 10 minutes). Lines show the mean, with error bars representing the standard deviation (n = 3). Statistically significant differences were determined by one-way ANOVA followed by Tukey’s HSD test (p < 0.05). Different letters indicate statistically different groups. (b) Mass spectrum showing mass changes of 200 µM RAP22-15 after 1 mM H2O2 treatment for 1 h then analysed by liquid chromatography mass spectrometry. The predominant peak ([M + H]+ = 1442.7 Da) represents unmodified RAP22-15, however ions likely representing RAP22-15 dimer ([M + 2H]2+ = 1441.7 Da) are also present, potentially arising from Nt-Cys-SOH and subsequent disulfide bond formation. Peaks at 1474.7 Da and 1490.7 Da represent Nt-Cys-SO2H and Nt-Cys-SO3H, respectively. (c) Tandem mass spectrometry confirms H2O2-mediated oxidative modification on Nt-Cys of RAP22-15; Spectrum shows b ions and y ions of fragmented 1474.7 Da peptide (Nt-Cys-SO2H) following H2O2 treatment RAP22-15 (collision energy 80 V). Expected y ions are observed, however b ions were predominantly observed with a consistent mass loss of 81 Da, termed b* ions. These ions likely correspond to loss of SO2 and NH3 upon fragmentation, as has been observed previously for an oxidative modification on N-terminal cysteine81. (d) Table showing the matched mass of the predicted fragments (b, b* and y) with the observed mass of RAP22-15 (1474.7 Da) under H2O2 treatment. (e) H2O2 dose-dependent effect on AtPCO1 enzyme activity (n = 3). (f) H2O2 dose-dependent effect on AtPCO2 enzyme activity (n = 3). For (e) and (f), 2 µM enzyme was treated with a series of H2 O2 concentrations (0–2 mM) at 4 °C for 30 minutes.
