Despite well-recognized biological importance, mass spectrometry (MS)-based glycomic identification of sulfo-, sialylated terminal glyco-epitopes on the N-glycans of various immune cell types remains technically challenging and rarely reported. Previous studies with monoclonal antibody have implicated a regulated expression of 6-sulfo-α2-6-sialyl LacNAc on B cells in peripheral lymph nodes and the circulating peripheral blood lymphocytes but its occurrence on leukemia cells or lymphomas have not been critically addressed. In this study, we have extended our previously developed MS-based sulfoglycomic platform by incorporating additional complementary analytical approaches in order to achieve a high sensitivity mapping and relative quantification of the detected sulfated glycotopes down to the level of defining their sialyl linkages. We showed that discovery mode sulfoglycomics and precise location of sulfate were best achieved by multimode MS analyses of fractionated, permethylated sulfated N-glycans. On the other hand, the relative degree of sulfation on individual N-glycans could be more efficiently inferred from the respective extracted ion chromatograms of native, non-sulfated and sulfated target N-glycans in single LC–MS/MS runs. The GlcNAc-6-O-sulfated α2-6-sialyl LacNAc, which constitutes the higher affinity ligand for the human inhibitory co-receptor of B cells, CD22, was found to be commonly carried on a range of complex type N-glycans from human CD19⁺ and CD4⁺ lymphocytes. We further showed that its occurrence on the most abundant α2-6-disialylated biantennary structure from the peripheral blood mononuclear cells of patients diagnosed as B-cell chronic lymphocytic leukemia varied within ±2-fold abundance from the mean value determined for isolated CD19+ lymphocytes and cultured B-CLL cells.

中文翻译：

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基于协同质谱的糖化学方法可精确定位人外周血单核细胞和淋巴细胞上的磺化唾液酸化N-聚糖

尽管生物学上的重要性已得到公认，但基于质谱（MS）的N上磺基，唾液酸化末端糖表位的糖基鉴定各种免疫细胞类型的β-聚糖在技术上仍然具有挑战性，鲜有报道。以前的单克隆抗体研究表明，6-硫代-α2-6-唾液酸LacNAc在外周淋巴结和循环外周血淋巴细胞的B细胞上表达受调控，但尚未明确解决其在白血病细胞或淋巴瘤上的发生。在这项研究中，我们通过合并其他互补的分析方法，扩展了我们先前开发的基于MS的硫糖代谢平台，从而实现了高灵敏度的定位和对检测到的硫酸化糖基的相对定量，直至定义其唾液酸键的水平。我们表明，通过分馏，全甲基化硫酸盐的多模式MS分析，可以最好地实现发现模式的糖酵解药和硫酸盐的精确定位N-聚糖。另一方面，从单个LC-MS / MS运行中的天然，未硫酸化和硫酸化的目标N聚糖的相应提取离子色谱图中，可以更有效地推断出各个N聚糖上的相对硫酸化程度。发现GlcNAc-6-O硫酸化的α2-6-唾液酸LacNAc构成了B细胞人类抑制性共受体CD22的更高亲和力配体，通常被携带在一系列复杂的N型聚糖中人CD19 ⁺和CD4 ⁺淋巴细胞。我们进一步表明，其发生在被诊断为B细胞慢性淋巴细胞性白血病的患者外周血单核细胞中最丰富的α2-6-二唾液酸化双天线结构上，其变化幅度与分离的CD19 +淋巴细胞和培养的B-CLL细胞。