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.

本研究的聚糖连接分析（Glycan linkage determination）由加州大学圣地亚哥分校聚糖分析核心中心（GlycoAnalytics Core center）提供技术服务完成。简言之，先采用碱-硼氢化物处理法进行还原性β消除，从经GtfAB、Nss及Gly糖基化的纯化GST-SRR1中释放聚糖。随后依照修库安努与凯雷克（Ciucannu and Kerek）的方法对O-聚糖（O-glycans）进行纯化与全甲基化修饰。具体操作如下：将O-聚糖冻干后溶于无水二甲基亚砜（anhydrous DMSO），依次加入二甲基亚砜中的氢氧化钠混悬液与碘甲烷。通过水-氯仿混合液进行两相萃取以获取全甲基化聚糖，收集氯仿层并冻干，用于后续的连接分析。将全甲基化聚糖置于4N三氟乙酸（4N TFA）中，于100℃下水解6小时，随后移除酸液，经氘代硼氢化钠（sodium borodeuteride）还原后进行乙酰化，得到全甲基化糖醇乙酸酯（PMAA）衍生物。最后采用搭载Restek-5ms毛细管柱（Restek-5ms capillary column）的气相色谱-质谱联用（GC-MS）系统对残基间连接进行分析，通过保留时间与电子轰击（EI）质谱碎裂模式的组合实现聚糖结构鉴定。