A Chemoenzymatic Approach Toward the Rapid and Sensitive Detection of O - GlcNAc Posttranslational Modifications - Supporting Information -

General Methods: Chemicals and reagents were used without further purification unless otherwise noted. If necessary, reactions were performed under argon atmosphere using anhydrous solvents. Thin layer chromatography was performed using E. Merck silica gel 60 F254 precoated plates and visualized using cerium ammonium molybdate stain. Flash column chromatography was carried out with Silica Gel 60 (230-400 mesh). NMR spectra were obtained on a Varian Mercury 300 instrument. High resolution mass spectra were obtained with a Jeol JMS-600H spectrometer. The peptide TAPTS(O-GlcNAc)TIAPG was synthesized at the Beckman Institute Biopolymer Synthesis Center using standard Fmoc chemistry. The Fmoc-protected, peracetylated O-GlcNAc serine amino acid was kindly synthesized by S. Tully as reported by Seitz et al. Baculovirus preparation and protein expression of CREB in Spodoptera frugiperda (Sf9) cells were performed by Dr. P. Snow at the Beckman Institute Protein Expression Facility at the California Institute of Technology. HeLa cell nuclear extracts were kindly prepared in H.-C. Tai according to published procedures. Y289L and wild-type GalT were expressed and purified as described previously. All protein concentrations were measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA).