HILIC and ERLIC Enrichment of Glycopeptides Derived from Breast and Brain Cancer Cells

Aberrant glycosylation has been linked to many different cancer types. The blood–brain barrier (BBB) is a region of the brain that regulates the entrance of ions, diseases, toxins, and so on. However, in breast cancer metastasis, the BBB fails to prevent the crossing of the cancer cells into the brain. Here we present a study of identifying and quantifying the glycosylation of six breast and brain cancer cell lines using hydrophilic interaction liquid chromatography (HILIC) and electrostatic repulsion liquid chromatography (ERLIC) enrichments and LC–MS/MS analysis. Qualitative and quantitative analyses of N-linked glycosylation were performed by both enrichment techniques for individual and complementary comparison. Potential cancer glycopeptide biomarkers were identified and confirmed by chemometric and statistical evaluations. A total of 497 glycopeptides were characterized, of which 401 were common glycopeptides (80.6% overlap) identified from both enrichment techniques. HILIC enrichment yielded 320 statistically significant glycopeptides in 231BR relative to the other cell lines out of 494 unique glycopeptides, and sequential HILIC-ERLIC enrichment yielded 214 statistically significant glycopeptides in 231BR compared with the other cell lines out of 404 unique glycopeptides. The results provide the first comprehensive glycopeptide listing for these six cell lines.异常糖基化与多种癌症类型密切相关。血脑屏障（blood–brain barrier, BBB）是大脑中调控离子、病原体、毒素等物质进入脑组织的区域。然而在乳腺癌转移过程中，血脑屏障无法阻止癌细胞向脑内侵袭。本研究针对6株乳腺癌及脑癌细胞系，采用亲水作用液相色谱（hydrophilic interaction liquid chromatography, HILIC）与静电排斥液相色谱（electrostatic repulsion liquid chromatography, ERLIC）富集结合液相色谱-串联质谱（LC-MS/MS）分析的方法，开展糖基化的鉴定与定量研究。分别采用两种富集技术对N-连接糖基化进行定性与定量分析，以实现独立检测与互补对照。通过化学计量学与统计学评估，鉴定并验证了潜在的癌症糖肽生物标志物。本研究共鉴定得到497条糖肽，其中401条为两种富集技术共同检出的共有糖肽（重叠率达80.6%）。在494条独特糖肽中，HILIC富集技术可在231BR细胞系中筛选出相较于其他细胞系具有统计学差异的320条糖肽；而在404条独特糖肽中，先HILIC后ERLIC的序贯富集策略可在231BR细胞系中筛选出相较于其他细胞系具有统计学差异的214条糖肽。本研究结果为上述6株细胞系提供了首个全面的糖肽列表。