The analysis of intact glycopeptides is a challenge because of the structural variety of the complex conjugates. In this work, we used separation involving hydrophilic interaction liquid chromatography using a superficially porous particle HALO® penta-HILIC column with tandem mass spectrometric detection for the analysis of N-glycopeptides of hemopexin. We tested the effect of the mobile phase composition on retention and separation of the glycopeptides. The results indicated that the retention of the glycopeptides was the combination of partitioning and adsorption processes. Under the optimized conditions, our HILIC method showed the ability to efficiently separate the glycoforms of the same peptide backbone including separation of the isobaric glycoforms. We achieved efficient separation of core and outer arm linked fucose of bi-antennary and tri-antennary glycoforms of the SWPAVGNCSSALR peptide and bi-antennary glycoform of the ALPQPQNVTSLLGCTH peptide, respectively. Moreover, we demonstrated the separation of antennary position of sialic acid linked via α2-6 linkage of the monosialylated glycopeptides. Glycopeptide isomers are often differentially associated with various biological processes. Therefore, chromatographic separation of the species without the need for an extensive sample preparation appears attractive for their identification, characterization, and reliable quantification.

中文翻译：

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亲水作用液相色谱法分离糖肽及其异构体

由于复杂缀合物的结构多样性，完整糖肽的分析是一项挑战。在这项工作中，我们使用了涉及亲水相互作用色谱的分离方法，该方法使用表面多孔颗粒HALO®penta-HILIC色谱柱和串联质谱检测技术进行N的分析-血红蛋白糖肽。我们测试了流动相组成对糖肽保留和分离的影响。结果表明，糖肽的保留是分配和吸附过程的结合。在优化的条件下，我们的HILIC方法显示了有效分离相同肽主链糖型的能力，包括分离同量糖型。我们分别实现了SWPAVGNCSSALR肽双天线和三天线糖型和ALPQPQNVTSLLGCTH肽双天线糖型的核心和外臂连接岩藻糖的有效分离。而且，我们证明了通过单唾液酸化糖肽的α2-6键连接的唾液酸触角位置的分离。糖肽异构体通常与各种生物学过程存在差异。因此，无需大量样品制备即可进行色谱分离，这对于它们的鉴定，表征和可靠的定量而言似乎很有吸引力。