Fig. 8: Identification of hyperreactive methionines through chemoproteomic profiling.

a Identification of hyperreactive methionine sites in human proteome using dose-dependent labeling (1-250 µM) with 1i probe. Excel sheet of analysis is included as Supplementary Data 2. b Heatmap visualization of 1i modified peptides to identify proteins with hyperreactive methionine residues. Cluster 11 was observed to possess 9 proteins with hyperreactive methionine sites with most of the modified proteins possessing enzymatic, regulatory, and structural functions. Centre line – median; box limits contain 50% of data; upper and lower quartiles, 75% and 25%; maximum – greatest value excluding outliers; minimum – least value excluding outliers; outliers – more than 1.5 times of the upper and lower quartiles, with n = 1 biological independent sample. c Functional category of the identified proteins shows a diverse class of proteins bearing hyperreactive methionines (10 µM) with a maximum labeling of enzymes/regulatory proteins. d Sequence analysis of the identified peptides (10 µM dose) demonstrate an overrepresentation of negatively charged residues near the labeled Met and underrepresentation of positively charged and aromatic residues showcasing that CuNiP targets oxidation-prone Met residues. e In-gel fluorescence analysis and profiling of oxidation-sensitive methionine residues. A dose-dependent labeling of methionine was observed with increasing H₂O₂ concentration, with the highest fluorescence intensity observed in control sample without H₂O₂ treatment. f Chemoproteomic profiling of oxidation sensitive methionine residues through treatment of T-47D cell lysate samples with H₂O₂, followed by subsequent treatment with probe 1i led to a dose-dependent decrease in normalized PSMs of 1i labeled peptides with increased H₂O₂ concentration. g dose-dependent decrease in peptide spectrum matches (PSMs) and intensity were observed as H₂O₂ concentration increases (0.5 mM of H₂O₂ 88 PSMs <control; 1 mM of H₂O₂ 177 PSMs <control; 0.5 mM of H₂O₂ 240 PSMs <control). The data utilized for analysis of figure (e, f, g) were acquired with n = 1 biological independent sample. Excel sheet of analysis is included as Supplementary Data 3. Source data are provided as a Source Data file. Figure 8, created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license” (Agreement number: BT26NXVCTX).