Figure 4.

Further characterization of chemoenzymatic mass tagging by SPAAC. (A) Commercial reagents are sufficient for O-GlcNAc quantitation. Cell lysates were subjected to the SpAAC mass tagging protocol using purified GalT or a commercially available enzyme (ThermoFisher Scientific) before Western blotting. (B) SPAAC proceeds selectively and quantitatively in 5 min at 98°C. OGlcNAcylated ubiquitin was added to H1299 lysates and enzymatically labeled. SPAAC was then performed at either room temperature for 16 h or 98°C for 5 min. Notably, we found that 98°C for 5 min results in quantitative labeling and does not increase the nonspecific background.