Alterations of protein glycosylation can serve as sensitive and specific disease biomarkers. Labeling procedures for improved separation and detectability of oligosaccharides have several drawbacks, including incomplete derivatization, side-products, noticeable desialylation/defucosylation, sample loss, and interference with downstream analyses. Here, we develop a label-free workflow based on high sensitivity capillary zone electrophoresis-mass spectrometry (CZE-MS) for profiling of native underivatized released N-glycans. Our workflow provides a >45-fold increase in signal intensity compared to the conventional CZE-MS approaches used for N-glycan analysis. Qualitative and quantitative N-glycan profiling of purified human serum IgG, bovine serum fetuin, bovine pancreas ribonuclease B, blood-derived extracellular vesicle isolates, and total plasma results in the detection of >250, >400, >150, >310, and >520 N-glycans, respectively, using injected amounts equivalent to <25 ng of model protein and nL-levels of plasma-derived samples. Compared to reported results for biological samples of similar amounts and complexity, the number of identified N-glycans is increased up to ~15-fold, enabling highly sensitive analysis of sample amounts as low as sub-0.2 nL of plasma volume equivalents. Furthermore, highly sialylated N-glycans are identified and structurally characterized, and untreated sialic acid-linkage isomers are resolved in a single CZE-MS analysis.

蛋白质糖基化的改变可以作为敏感且特异性的疾病生物标志物。用于改善寡糖分离和可检测性的标记程序有几个缺点，包括不完全衍生化、副产物、明显的脱唾液酸化/去岩藻糖基化、样品损失以及对下游分析的干扰。在这里，我们开发了一种基于高灵敏度毛细管区带电泳质谱 (CZE-MS) 的无标记工作流程，用于对天然未衍生化释放的 N-聚糖进行分析。与用于 N-聚糖分析的传统 CZE-MS 方法相比，我们的工作流程将信号强度提高了 >45 倍。对纯化人血清 IgG、牛血清胎球蛋白、牛胰腺核糖核酸酶 B、血源性细胞外囊泡分离物和总血浆进行定性和定量 N-聚糖分析，可检测 >250、>400、>150、>310 和>520 N-聚糖，分别使用相当于 <25 ng 模型蛋白和 nL 水平血浆来源样品的注射量。与相似数量和复杂性的生物样品的报告结果相比，已识别的 N-聚糖数量增加了约 15 倍，从而能够对低至血浆体积当量低于 0.2 nL 的样品量进行高度灵敏的分析。此外，高度唾液酸化的 N-聚糖被鉴定并进行结构表征，并且在单次 CZE-MS 分析中解析了未经处理的唾液酸连接异构体。