Linkage analysis of GspB glycosylated by GtfAB, Nss and Gly

Glycan linkage<b> </b>determination was carried out as service performed by the GlycoAnalytics Core center at the University of California, San Diego. In brief,<b> </b>Glycans were liberated from purified GST-SRR1 (glycosylated by GtfAB, Nss and Gly) by reductive beta elimination using base-borohydride treatment. The <i>O</i>-glycans were then purified and per-methylated according to the method of Ciucannu and Kerek. In brief, <i>O</i>-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.

本次聚糖连接位点分析由加州大学圣迭戈分校糖组学分析核心中心（GlycoAnalytics Core Center）提供技术服务完成。简言之，先通过碱-硼氢化物还原β消除法，从经GtfAB、Nss与Gly糖基化修饰的纯化GST-SRR1蛋白中释放聚糖。随后根据Ciucannu与Kerek建立的方法，对所得O-聚糖（O-glycan）进行纯化与全甲基化修饰。具体操作为：将O-聚糖干燥后溶于无水二甲基亚砜（DMSO），随后加入二甲基亚砜体系中的氢氧化钠混悬液与碘甲烷。通过水-氯仿混合液萃取分离得到全甲基化聚糖，收集氯仿层并干燥后用于连接位点分析。将全甲基化聚糖置于4N三氟乙酸（TFA）中，于100℃水解6小时，随后移除酸试剂，以氘代硼氢化钠进行还原，再经乙酰化处理得到全甲基化糖醇乙酸酯（per-methylated alditol acetate, PMAA）衍生物；随后采用搭载Restek-5ms毛细管柱的气相色谱-质谱联用仪（GC-MS）开展残基间连接位点分析。化合物鉴定通过保留时间与电子轰击（EI）质谱碎裂图谱的联合比对完成。