Linkage analysis of GspB glycosylated by GtfAB, Nss and Gly

Glycan linkage determination was carried out as service performed by the GlycoAnalytics Core center at the University of California, San Diego. In brief, Glycans were liberated from purified GST-SRR1 (glycosylated by GtfAB, Nss and Gly) by reductive beta elimination using base-borohydride treatment. The O-glycans were then purified and per-methylated according to the method of Ciucannu and Kerek. In brief, O-glycans were dried and dissolved in anhydrous DMSO, followed by adding NaOH slurry in DMSO and methyl iodide. Per-methylated glycans were extracted by partitioning in water-chloroform mixture and the chloroform layer was dried and used for linkage analysis. The per-methylated glycans were hydrolyzed using 4N TFA at 100°C for 6 hr, followed by removal of the acid, reduced with sodium borodeuteride and acetylated to generate the per-methylated alditol acetate (PMAA) derivative, which was then analyzed by GC-MS using Restek-5ms capillary column for inter-residue linkages. Identifications are achieved by using a combination of retention times and EI mass fragmentation pattern.