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Profiling of N-linked glycans from 100 cells by capillary electrophoresis with large-volume dual preconcentration by isotachophoresis and stacking
Journal of Chromatography A ( IF 4.1 ) Pub Date : 2018-06-23 , DOI: 10.1016/j.chroma.2018.06.034
Takayuki Kawai , Nobutoshi Ota , Akiko Imasato , Yoko Shirasaki , Koji Otsuka , Yo Tanaka

Glycan structure is changed in response with pathogenesis like cancer. Profiling of glycans from limited number of pathogenetic cells in an early-stage tissue is essential for discovering effective drugs. For analyzing tiny biological samples, we developed sensitive, high-resolution, and salt-tolerant method for analyzing trace level of N-linked glycans by coupling capillary electrophoresis (CE), laser-induced fluorescence (LIF) detection, and a new online sample preconcentration (OSP) method named “large-volume dual preconcentration by isotachophoresis and stacking (LDIS)”, which is composed of two OSP methods, large-volume sample stacking (LVSS) and transient isotachophoresis (tITP). A typical LDIS-CE-LIF protocol was simple: a short-plug of leading electrolyte (LE) and large-volume sample solution were introduced to a capillary, followed by application of constant voltage. In the analysis of glucose ladder labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 10 mM sodium chloride as LE, up to 2300-fold sensitivity increase was achieved with higher resolution than those in normal CE. By applying pressure assist during preconcentration, both viscous gel electrolyte and salty matrix of up to 10 mM NaCl were acceptable. Finally, N-glycans from approximately 100 cells (HeLa, MCF7, and HepG2) were analyzed as the model of localized tumor cells. From 30 to 40 glycans were successfully detected with almost same profile of large-scale sample. N-glycan structure could be predicted by searching glucose-unit value via Glycobase database, indicating that HepG2 expressed more sialylated glycans and MCF-7 expressed less glycans respectively, comparing with HeLa cells. It suggests the potential of LDIS-CE-LIF for discovery of disease-specific N-linked glycans in microscale environment.



中文翻译:

通过毛细管电泳从等体积电泳和堆叠进行大体积双重预浓缩从100个细胞中分离出N-连接的聚糖

糖基结构因癌症等发病机制而发生变化。在早期组织中从数量有限的病原体细胞中进行聚糖分析对于发现有效的药物至关重要。为了分析微小的生物样品,我们开发了灵敏,高分辨率和耐盐的方法来分析痕量N毛细管电泳(CE),激光诱导荧光(LIF)检测和一种新的在线样品预浓缩(OSP)方法(称为“等速电泳和堆叠大体积双重预浓缩”(LDIS))组成的链状聚糖两种OSP方法是大体积样品堆积(LVSS)和瞬时等速电泳(tITP)。典型的LDIS-CE-LIF协议很简单:将短引线的领先电解质(LE)和大体积样品溶液引入毛细管,然后施加恒定电压。在分析以10 mM氯化钠为LE的8-氨基py-1,3,6-三磺酸标记的葡萄糖梯子时,与普通CE相比,分辨率提高了2300倍。通过在预浓缩过程中施加压力辅助,粘性凝胶电解质和最高达10 mM NaCl的咸性基质都是可以接受的。最后,分析了来自大约100个细胞(HeLa,MCF7和HepG2)的N-聚糖,作为局部肿瘤细胞的模型。成功检测到30至40个聚糖,并且具有与大型样品几乎相同的轮廓。N-聚糖结构可通过在Glycobase数据库中搜索葡萄糖单位值来预测,表明与HeLa细胞相比,HepG2分别表达更多的唾液酸化聚糖和MCF-7表达更少的聚糖。它暗示了LDIS-CE-LIF在微观环境中发现疾病特异性N-连接聚糖的潜力。

更新日期:2018-06-23
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