Glycoprofile β elimination kit

In vitro O-GlcNAcylation of TAB3 was detected as previously described [8 (link)]. The TAB3 (1 mM) was added into 20 μl reaction buffer (50 mMTris-HCl [pH = 7.5], 1 mM DTT, 12.5 mM MgCl₂) which contain 50 mM OGT and 1 mM UDP-GlcNAc. The reaction mixtures were incubated for 90 min at 37°C, stopped by adding loading buffer, resolved on Western blot with appropriate antibodies. To enzymatic label TAB3 at O-GlcNAc site, O-GlcNAcylated GST-TAB3 bound beads and was labeled using Click-iT^™ O-GlcNAc enzymatic labeling system (Invitrogen, Carlsbad, USA) and detected by Click-iT^™ biotin protein analysis detection kit (Invitrogen, Carlsbad, USA) following manufacturer's recommendation. β-elimination was performed overnight at 4°C using the GlycoProfile β-elimination kit (Sigma, St. Louis, USA) according to the manufacturer's instructions.

To confirm O-GlcNAc sites an on-resin dephosphorylation step between the on-resin proteolytic digest and the on-resin β-elimination was added. So ideally all peptides bound to the alkyne resin should be O-GlcNAc modified. O-GlcNAcylated peptides linked to agarose beads were dephosphorylated at 37°C for 6 h in 400 μL of dephosphorylation buffer using 800 U of λ phosphatase and 20 U of calf intestine phosphatase. After dephosphorylation, the resin was washed twice with 1.5 mL of water and the slurry volume was adjusted to 300 μL with water before treatment with the GlycoProfile β-elimination Kit (Sigma Aldrich). The reaction mixture was incubated in an end-over-end shaker with extensive mixing at 4°C and quenched after 24 h with 1% trifluoroacetic acid (TFA). Agarose beads were discarded, and the solution containing β-eliminated peptides, corresponding to O-GlcNAcylated peptides, was desalted in C18 reversed-phase columns and dried in a centrifugal vacuum system before LC-MS/MS analysis.