Figure 2.

DJ-1 cannot remove stable AGEs from proteins. (A) To assess the potential deglycase activity of DJ-1, two sets of conditions were evaluated. In the concurrent treatment, DJ-1 was co-incubated with both ubiquitin (Ub) and MGO for 24 h at 37 °C in 20 mM PBS at pH 7.3. In the subsequent treatment, Ub was first glycated by MGO for 24 h at 37 °C in 20 mM PBS at pH 7.3, at which point the excess MGO was quenched with Tris buffer (see also Figure S2). Afterward, DJ-1 was incubated with the glycated protein for an additional 24 h at 37 °C in 20 mM PBS and 15 mM Tris at pH 7.3. (B) Using SDS-PAGE and western blotting against MGO, it was possible to observe a decrease in glycation from concurrent treatments with wild-type DJ-1 (DJ-1^WT), but not a catalytically inactive DJ-1 variant (DJ-1^C106A). (C) These same findings were obtained using intact protein mass spectrometry following subsequent or concurrent DJ-1 treatments, as observed in the representative deconvoluted mass spectra.