Biomedical Omics Group, Korea Basic Science Institute, Cheongju, Republic of Korea.
Biomedical Omics Group, Korea Basic Science Institute, Cheongju, Republic of Korea; Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Republic of Korea.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Mar 15;1110-1111:101-107. doi: 10.1016/j.jchromb.2019.02.015. Epub 2019 Feb 13.
Analyses of intact glycopeptides using mass spectrometry is challenging due to the numerous types of isomers of glycan moieties attached to the peptide backbone. Here, we demonstrate that high-temperature reversed-phase liquid chromatography (RPLC) can be used to separate isomeric O- and N-linked glycopeptides. In general, high column temperatures enhanced the resolution for separation of sialylated O- and N-linked glycopeptide isomers with decreased retention times. Using the high-temperature RPLC method, α2-6-linked sialylated N-glycopeptides were eluted first, followed by α2-3-linked isomers. However, highly sialylated N-glycopeptides containing hydrophobic amino acids exhibited increased retention times at high temperature. The separation of sialylated O- and N-glycopeptides with different glycan isoforms using a high-temperature RPLC method was demonstrated. This study indicates that reversed-phase chromatographic separation at high column temperatures is suitable for the separation of glycopeptide structural isomers.
由于与肽主链相连的糖基部分的异构体种类繁多，因此使用质谱法分析完整的糖肽具有挑战性。在这里，我们证明高温反相液相色谱（RPLC）可用于分离异构的 O-和 N-连接的糖肽。通常，较高的柱温会缩短保留时间，从而增强了对唾液酸化的 O-和 N-连接糖肽异构体的分离分辨率。使用高温 RPLC 方法，首先洗脱α2-6 连接的唾液酸化 N-糖肽，然后是α2-3 异构体。但是，含有疏水性氨基酸的高度唾液酸化的 N-糖肽在高温下表现出增加的保留时间。使用高温 RPLC 方法证明了不同糖型的唾液酸化 O-和 N-糖肽的分离。这项研究表明，在高温下进行反相色谱分离适用于糖肽结构异构体的分离。
