Related Experiment Video
Updated: Jan 28, 2026

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides
Published on: November 2, 2021
Isomer separation of sialylated O- and N-linked glycopeptides using reversed-phase LC-MS/MS at high temperature
Eun Sun Ji1, Hyun Kyoung Lee2, Gun Wook Park1
1Biomedical Omics Group, Korea Basic Science Institute, Cheongju, Republic of Korea.
Abstract:
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.
Related Concept Videos
Constitutional Isomers of Alkanes
The linear isomer of an alkane is prefixed by the term “n”; hence a linear isomer of pentane is known as n-pentane. Based on the type of branching, some of the...
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein....
Phase Diagrams
Phase Transitions: Melting and Freezing
Phase Transitions: Sublimation and Deposition
Phase Transitions

