Identification of differentially expressed proteins by treatment with PUGNA c in 3 T 3‐ L 1 adipocytes through analysis of ATP ‐binding proteome

O ‐ G lc NA c (2‐acetamino‐2‐deoxy‐β‐ D ‐glucopyranose), an important modification for cellular processes, is catalyzed by O ‐ G lc NA c transferase and O ‐ G lc NA case. O ‐(2‐acetamido‐2‐deoxy‐ D ‐glucopyranosylidene) amino‐ N ‐phenylcarbamate ( PUGNA c) is a nonselective inhibitor of O ‐ G lc NA case, which increases the level of protein O ‐ G lc NA cylation and is known to induce insulin‐resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNA c. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3 T 3‐ L 1 adipocytes and C 2 C 12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP ‐bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP ‐binding protein families classified by PROSITE . The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3 T 3‐ L 1 adipocytes following treatment with PUGNA c. For label‐free quantitation, a gel‐assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data‐independent (671 proteins identified) and data‐dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide‐binding proteins and we focused on some nucleotide‐binding proteins, ubiquitin‐activation enzyme 1 ( E 1), H sp70, vasolin‐containing protein ( V cp), and H sp90, involved in ubiquitin‐proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNA c resulted in increased ubiquitination of proteins in a time‐dependent manner, and a decrease in both the amount of A kt and the level of phosphorylation of A kt, a key component in insulin signaling, through downregulation of H sp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNA c. This result would provide insight into understanding functions of PUGNA c in 3 T 3‐ L 1 cells.