Figure 5.

Dynamics of protein S-alkylation by aHNE in RKO cells. (A) Heatmap of ratios of changes of all detected cysteine S-alkylation events shows that most adducts turn over rapidly in a time-dependent manner in cells. Lower the measured ratio (L/H) indicates more rapid turnover. (B) XIC are shown for changes in S-alkylated peptides from FAM120A protein in RKO cells, with the profiles for light- and heavy- labeled peptides in red and blue, respectively. The mean measured ratios were calculated from three biological replicate experiments and are displayed below the individual chromatograms, respectively. (C) Turnover of alkylation is not affected by proteasome inhibition. RKO cells were pretreated with (red) or without MG132 (black), followed by aHNE treatment with or without 1 and 4 h recovery periods. Proteins alkylated by aHNE were labeled with azido-biotin and detected by Western blotting with fluorescein-conjugated streptavidin. Data were presented as mean values ± SD, n = 3 biological replicates per group. A representative Western blot is shown in Figure S10 in the Supporting Information. (D) Distributions of the measured ratios of dynamic aHNE-cysteine adduction in vitro (red) and in situ (white).