A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn
A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn
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DOI：
10.1093/glycob/cwp048
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发表时间：
2009-07-01
期刊：
影响因子：
4.3
通讯作者：
Hansson, Gunnar C.
中科院分区：
文献类型：
--
作者：
Larsson, Jessica M. Holmen;Karlsson, Hasse;Hansson, Gunnar C.
高灵敏度的聚糖谱分析提供详细的结构信息是非常重要的，在寻找聚糖疾病的标志物。通过将粘蛋白的直接和快速制备方案与高通量nanoLC/MS相结合，我们已经能够研究在25名正常对照患者的常规结肠镜检查期间从乙状结肠收集的单次活检（类似于5 mg湿组织作为起始材料）的结肠MUC 2粘蛋白的O-糖基化。从氯化胍提取的不溶性沉淀中回收这种大的粘蛋白糖蛋白，还原并烷基化，通过SDS-琼脂糖聚丙烯酰胺复合凝胶电泳分离，并转移到PVDF膜上。通过还原性β消除释放主要MUC 2单体条带的O-连接低聚糖，并通过nanoLC/质谱法和MSn进行分析。目的是鉴定乙状结肠的MUC 2 O-聚糖，并提供O-聚糖库的全面目录。鉴定了100多种复杂的O-连接寡糖，其中一些以前没有描述过。大多数寡糖基于核心3结构，在GalNAc的6位具有唾液酸，并且在大多数聚糖中发现了子结构Gal β 1-3/4-GlcNAc β 1-3（NeuAc-6）GalNAcol。最丰富的组分是SO 3--Gal-（Fuc）GlcNAc-3（NeuAc-6）GalNAcol、GalNAc-（NeuAc-）Gal-4/3GlcNAc-3（NeuAc-6）GalNAcol、GalNAc-3（NeuAc-6）GalNAcol和GlcNAc-3（NeuAc-6）GalNAcol。与其他粘蛋白的O-聚糖相反，正常个体中的S形MUC 2 O-聚糖库和相对量相对恒定。
High-sensitivity glycan profiling providing detailed structural information is very important in the search for glycan disease markers. By combining a straight-forward and fast preparation protocol of mucins with high-throughput nanoLC/MS, we have been able to study the O-glycosylation of the colon MUC2 mucin from one single biopsy (similar to 5 mg wet tissue as starting material) collected from the sigmoid colon during routine colonoscopy of 25 normal control patients. This large mucin glycoprotein was recovered from the guanidinium chloride-extracted insoluble pellet, reduced and alkylated, separated by SDS-agarose polyacrylamide composite gel electrophoresis, and transferred to a PVDF membrane. The O-linked oligosaccharides of the major MUC2 monomer band were released by reductive beta-elimination and analyzed by nanoLC/mass spectrometry and MSn. The aim was to identify the MUC2 O-glycans of the sigmoid colon and provide a comprehensive catalog of the O-glycan repertoire. More than 100 complex O-linked oligosaccharides were identified, of which some had not been described before. Most of the oligosaccharides were based on the core 3 structure with sialic acid at the 6-position of the GalNAc and the substructure Gal beta 1-3/4-GlcNAc beta 1-3(NeuAc-6)GalNAcol was found in most glycans. The most abundant components were SO3--Gal-(Fuc)GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-(NeuAc-)Gal-4/3GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-3(NeuAc-6) GalNAcol, and GlcNAc-3(NeuAc-6)GalNAcol. In contrast to the O-glycans of other mucins, the sigmoid MUC2 O-glycan repertoire and relative amounts in normal individuals were relatively constant.