Fig. 2.

Light- and ¹O₂-dependent Trp643 oxidation occurs in vivo. a EX1-GFP protein enrichment. For input, total proteins extracted from 5-day-old dark (DD)-grown or CL-grown seedlings of EX1-GFP ex1 were analyzed. For IP, immune-reactive EX1-GFP proteins were precipitated from each input sample by adding magnetic agarose beads conjugated with GFP antibody. Trapped proteins were solubilized and separated by SDS-PAGE and probed on western blots using the indicated antibodies. b Oxidative PTM (Oxi-PTM) analysis. The IP samples were subjected to PTM analysis by mass spectrometry (MS). EX1 was found to undergo light- and ¹O₂-dependent oxidation, especially at Trp643 in peptide ⁶⁴³WVDGELVILDGK⁶⁵⁴. This oxidation led to the formation of oxindolylalanine (OIA), N-formylkyrnurenine (NFK), and kynurenine (KYN) with + 16, + 32, and + 4 mass shifts, respectively. c The effect of increasing levels of ¹O₂ on Trp643 oxidation. To increase the levels of ¹O₂, 5-day-old seedlings of EX1-GFP ex1 flu grown under CL were subjected to 2 h dark and then re-exposed to light for 5 min (D/L). The GFP-trapped EX1-GFP proteins were subjected to MS analysis and the relative levels of Trp643 oxidation was measured. Oxidation levels were calculated using the sum of absolute intensities of the peptides carrying oxidized Trp643. Bar chart represents the relative intensity of the oxidized peptides. Asterisks indicate statistically significant differences between the mean values (P < 0.05, Student’s t-test)