Loss of endogenous DJ-1 had no effect on α-oxoaldehyde modifications but increased phospho-glyceroyl modifications. (A) Cell lysates from WT, DJ-1 KO, or DJ-1 KO HeLa cells expressing exogenous WT DJ-1 were supplemented with cPGA. Phospho-glyceroyl modifications were analyzed by Phos-tag SDS-PAGE, followed by immunoblotting with an anti-GAPDH antibody. (B) The same lysates were treated with 2 mM MGO and immunoblotted with an anti-MGO antibody. The cells without exogenous DJ-1 expression are denoted as none. Blots are representative of at least two independent experiments (A and B). (C) Schematic overview of the experimental setup to assess the phosphoglycerate and α-oxoaldehyde modifications. (D) Volcano plot of the log₂ fold change in 3-phosphoglyceroyl lysine peptide and the log₁₀ of the P values (Student’s t test). Peptides with 3-phosphoglyceroyl lysine that significantly increased or decreased in DJ-1 KO cells compared with WT cells (log₂ fold change > 1 or log₂ fold change less-than −1, P < 0.05) are shown in red (increase) and blue (decrease) circles, respectively. Mean fold changes and P values were calculated from three biological replicates (n = 3). (E) Volcano plot for CML and CEL peptides prepared as in D. (F) Abundance of CEL peptides in WT cells following treatment with MGO. Box plots show the distribution of values. Blue dots indicate individual data points, while gray dots represent outliers (n = 2). (G) Abundance of CML peptides in WT cells treated with glyoxal. Plots were drawn as in F (n = 2). Source data are available for this figure: SourceData F9.