Donczo Boglarka, Szarka Mate, Tovari Jozsef, Ostoros Gyorgyi, Csanky Eszter, Guttman Andras
Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Hungary.
National Institute of Oncology, Budapest, Hungary.
Electrophoresis. 2017 Jun;38(12):1602-1608. doi: 10.1002/elps.201600558. Epub 2017 Mar 23.
Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues.
采用激光诱导荧光检测的毛细管电泳（CE-LIF）技术，对来自甲醛固定石蜡包埋（FFPE）的小鼠肺、脑、心、脾、肝、肾和肠组织样本中释放的内切糖苷酶和荧光团标记的N-聚糖进行分析。首先对FFPE样本进行脱石蜡处理，随后进行溶解和糖蛋白回收。PNGase F介导N-连接寡糖的释放，然后用氨基芘三磺酸盐进行标记。在对FFPE小鼠组织进行CE-LIF糖谱分析后，通过基于外切糖苷酶阵列的碳水化合物测序对肺标本的N-聚糖库进行进一步研究。寡糖的结构归属借助GUcal软件和相关数据库，根据用相应外切糖苷酶反应混合物处理后的迁移率变化来完成。从小鼠肺FFPE组织糖组中测序并鉴定出16种主要的N-连接碳水化合物结构，即高甘露糖型（3种）、中性双天线型（3种）、唾液酸化单天线型（1种）和唾液酸化双天线型（9种）寡糖。后两种中的两种还含有α(1-3)连接的半乳糖残基。
