Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague 2, Czech Republic.
Department of Oncology, Lombardi Comprehensive Cancer Center PSB GF9, Georgetown University, 3800 Reservoir Road NW, Washington, DC, 20057, USA.
Anal Bioanal Chem. 2018 Aug;410(20):5001-5008. doi: 10.1007/s00216-018-1150-3. Epub 2018 May 28.
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.
完整糖肽的分析具有挑战性，因为复杂缀合物的结构种类繁多。在这项工作中，我们使用亲水相互作用液相色谱法，使用具有串联质谱检测的表面多孔颗粒 HALO® penta-HILIC 柱来分析血色素结合蛋白的 N-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 方法表现出有效分离同一肽主链的糖型的能力，包括分离等质异位的糖型。我们成功地分离了 SWPAVGNCSSALR 肽的双触角和三触角糖型以及 ALPQPQNVTSLLGCTH 肽的双触角糖型的核心和外臂连接的岩藻糖，分别。此外，我们还证明了通过α2-6 键连接的单唾液酸化糖肽中天冬氨酸连接位置的分离。糖肽异构体通常与各种生物过程相关。因此，无需广泛的样品制备即可分离这些物质，这对于它们的鉴定、表征和可靠定量似乎很有吸引力。
