RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, 565-0874, Japan; Japan Science and Technology Agency, PRESTO, Kawaguchi, Saitama, 332-0012, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan.
RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, 565-0874, Japan.
J Chromatogr A. 2018 Aug 31;1565:138-144. doi: 10.1016/j.chroma.2018.06.034. Epub 2018 Jun 23.
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.
聚糖结构会随着癌症等疾病的发生而发生变化。在早期组织中从有限数量的病态细胞中对聚糖进行分析对于发现有效药物至关重要。为了分析微小的生物样本，我们开发了一种灵敏、高分辨率、耐盐的方法，通过将毛细管电泳（CE）、激光诱导荧光（LIF）检测与一种新的在线样品预浓缩（OSP）方法“通过等速电泳和堆积的大体积双预浓缩（LDIS）”相结合，来分析痕量水平的 N-连接聚糖。LDIS-CE-LIF 协议非常简单：将短塞的先导电解质（LE）和大容量样品溶液引入毛细管，然后施加恒压。在分析用 8-氨基芘-1,3,6-三磺酸标记的葡萄糖阶梯作为 LE 的实验中，与正常 CE 相比，分辨率更高，灵敏度提高了 2300 倍。通过在预浓缩过程中施加压力辅助，可以接受高达 10mM NaCl 的粘性凝胶电解质和咸基质。最后，以局部肿瘤细胞的模型分析了大约 100 个细胞（HeLa、MCF7 和 HepG2）的 N-聚糖。从 30 到 40 种聚糖可以成功检测到，并且与大样本的图谱几乎相同。通过在 Glycobase 数据库中搜索葡萄糖单位值可以预测 N-聚糖结构，表明与 HeLa 细胞相比，HepG2 表达了更多的唾液酸化聚糖，而 MCF-7 表达了更少的聚糖。这表明 LDIS-CE-LIF 具有在微环境中发现疾病特异性 N-连接聚糖的潜力。
