High performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line MSn fragmentation to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MSn method development to resolve structural isomers on-the-fly. We report baseline separation and MSn fragmentation of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li+, which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations, robust structural characterization, and is amenable to auto-sampling with associated throughput enhancements.

中文翻译：

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反相nanoLC-NSI-MSn分离和鉴定过甲基化的聚糖异构体

数十年来，高效液相色谱已被用于增强生物混合物中复杂分析物的检测灵敏度和定量。在这些分析物中，从糖蛋白和糖脂释放的聚糖已通过与各种检测方法结合的HPLC表征为未衍生化或荧光标记的衍生物。这些方法已被证明对于分析糖蛋白和糖脂糖基化的结构多样性极为有用，但需要使用聚糖标准品和二级正交降解策略来验证结构分配。高效液相色谱法分离聚糖（作为其全甲基化衍生物）的可靠方法，加上在线MSn裂解，可确定独立于标准品的结构特征，将大大提高从生物样品中获得的知识深度。在这里，我们报告了用于LC-MS分析全甲基化聚糖的优化工作流程，其中包括样品制备，流动相优化和MSn方法开发以即时解决结构异构体。我们报告了基线分离和异构N-和O-聚糖结构的MSn片段化，辅以Li +补充流动相，这简化了加合物异质性并促进了交叉环片段化，从而获得了有价值的单糖键合信息。我们的工作流程已从基于蛋白质组学的标准工作流程改编而来，因此，为蛋白质组学方面的专门知识的实验室提供了机会，可在不偏离现有缓冲系统，色谱介质和仪器配置的情况下，获取糖类数据。此外，我们的工作流程不需要具有高分辨率/精确质量功能的质谱仪。对聚糖对人类健康和疾病的生物学意义的迅速发展的认识要求实施高通量方法，以鉴定和量化从足够大小的样本集中收获的聚糖，以实现适当的有力的统计意义。我们报告的LC-MSn方法可产生聚糖异构体分离，稳健的结构表征，并且可进行自动采样并具有相关的通量提高。对聚糖对人类健康和疾病的生物学意义的迅速发展的认识要求实施高通量方法，以鉴定和量化从足够大小的样本集中收获的聚糖，以实现适当的有力的统计意义。我们报告的LC-MSn方法可产生聚糖异构体分离，稳健的结构表征，并且可以进行自动采样并具有相关的通量提高。对聚糖对人类健康和疾病的生物学意义的迅速发展的认识要求实施高通量方法，以鉴定和量化从足够大小的样本集中收获的聚糖，以实现适当的有力的统计意义。我们报告的LC-MSn方法可产生聚糖异构体分离，稳健的结构表征，并且可以进行自动采样并具有相关的通量提高。