Mechanism of N-glycan Analysis Using HILIC-UHPLC

N-glycosylation is a crucial post-translational modification of proteins, widely present in eukaryotic cells. It plays an important role in various biological processes, such as protein folding, stability, cell recognition, and signal transduction. Therefore, accurate analysis of protein N-glycosylation is essential for understanding many biological phenomena and disease mechanisms. N-glycan analysis involves the separation, identification, and quantification of glycan structures. However, the polarity and structural complexity of glycans pose significant challenges for analysis. HILIC-UHPLC (Hydrophilic Interaction Liquid Chromatography-Ultra High Performance Liquid Chromatography) has proven to be an effective technique for the separation and analysis of glycans, enabling accurate analysis of N-glycans in complex biological samples.

 

Working Principle of HILIC-UHPLC

HILIC-UHPLC is a liquid chromatography technique based on hydrophilic interactions and is particularly suitable for the separation of polar molecules. Its core mechanism involves exploiting the interactions between polar stationary phases and polar mobile phases, causing variations in retention times of the analytes on the stationary phase, thereby achieving separation. In N-glycan analysis, HILIC-UHPLC separates different types of glycans through hydrophilic interactions between polar molecules.

 

1. Selection of Stationary and Mobile Phases

The separation efficiency of HILIC-UHPLC depends on the choice of stationary and mobile phases. Typically, the stationary phase consists of highly polar materials, such as silica or hydroxyl compounds, which can form strong interactions with the polar groups on glycans. The mobile phase usually consists of a polar solution with a high concentration of organic solvent (e.g., acetonitrile). By adjusting the proportion of organic solvent, the retention time of molecules on the stationary phase can be altered, enabling the separation of different glycans.

 

2. Separation Mechanism

In HILIC-UHPLC, the separation of N-glycans primarily relies on their polarity. Larger glycans contain more polar groups, leading to stronger interactions with the stationary phase and longer retention times, whereas smaller glycans have shorter retention times. By adjusting the polarity gradient of the mobile phase, smaller glycans are eluted first, followed by larger glycans, achieving separation.

 

Additionally, the stationary phase in HILIC can selectively separate glycans based on structural specificity. For example, different terminal sugar residues, such as fucose and galactose, may influence the strength of the interactions between glycans and the stationary phase, further optimizing the separation.

 

Advantages of HILIC-UHPLC in N-Glycan Analysis

Compared to traditional analytical techniques, HILIC-UHPLC offers significant advantages in N-glycan analysis. First, it achieves efficient separation without damaging glycan structures. Second, it is suitable for analyzing various polar molecules in samples, especially highly polar or structurally complex glycans. Additionally, HILIC-UHPLC, with its excellent separation capabilities and high sensitivity, can be coupled with mass spectrometry (MS) to further enhance the quantification and identification of N-glycans.

 

HILIC-UHPLC is an efficient tool for analyzing N-glycans, utilizing hydrophilic interaction-based separation mechanisms to achieve high-performance and sensitive glycan analysis in complex biological samples.