Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC–MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.

蛋白质糖基化的分析是一项挑战，需要正交方法才能实现多种糖型的分离。反相色谱与串联质谱联用（RP-LC-MS / MS）是糖肽分析最强大的工具之一。在这项工作中，我们在与MS / MS结合的芯片上开发并比较了纳米级的RP-LC和亲水相互作用液相色谱（HILIC），以分离从血红素的胰蛋白酶消化物中得到的两种肽的糖型。我们观察到保留时间的减少是由于HILIC中连接至相同肽主链的聚糖极性降低。对于RP-LC观察到相反的效果。唾液酸的存在延长了两种色谱模式下糖肽的保留时间。与nanoRP-LC相比，基于聚糖的成分，nanoHILIC方法提供了更高的选择性，但灵敏度较低。nanoHILIC方法能够将SWPAVGDCSSALR糖肽双触角糖型上的岩藻糖（核心和外臂）的连接异构体部分分离，这有助于阐明岩藻糖基化糖型的结构。