HILIC-Based Breakthroughs for Glycopeptides and Glycans

In a study published in the Journal of Chromatography A, led by scientists from Soochow University in Suzhou, China, hydrophilic interaction liquid chromatography (HILIC) breakthroughs were reviewed in relation to glycopeptide enrichment and glycan separation (1).

Binary pump in HPLC system. High performance liquid chromatography at analytical chemistry laboratory. | Image Credit: © vladim_ka - stock.adobe.com

A glycan is a compound that consists of several monosaccharides, either in free form or attached to a different substance (2). Living systems rely on these substances, alongside glycoRNAs and glycolipids for crucial functions in different biochemical processes. Glycans also play a role in forming the extracellular matrix, which is vital for cell growth, differentiation, and maintaining tissue structure. The variety of glycan structures and their differential expression across different cell types and tissues provides basis for cell identification and communication.

Protein glycosylation is a vital posttranslational modification initiated in the endoplasmic reticulum, a large and dynamic structure that serves many roles in a cell, including calcium storage, protein synthesis, and lipid metabolism (3,4). However, it currently faces analytical challenges, mainly due to heterogeneous glycosite, diverse glycans, and mass spectrometry (MS) limitations.

In this review, the scientists summarized recent breakthroughs in hydrophilic interaction liquid chromatography (HILIC). HILIC is a high-performance liquid chromatographic (HPLC) technique for separating polar and hydrophilic compounds, being a normal-phase type of separation that uses reversed-phase type eluents. This process provides a column with a hydrophilic stationary phase and an eluent with water, buffer and a high concentration of water-miscible organic solvent (5). HILIC has seen recent advancements in different stationary phases, such as Amide-80, glycoHILIC, amino acids, or peptides, for improved HILIC-based glycopeptide analysis.

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Numerous advancements in HILIC materials for glycopeptide enrichment and glycan separation have been made, offering new and exciting prospects for understanding glycoproteomics and glycobiology, the study of glycans attached to proteins, and their biological roles. For improving selectivity and binding efficiency, designing HILIC materials with intricate sugar combinations or integrating diverse hydrophilic functional groups with advanced materials shows promise. Though capturing specific glycans, namely O-linked and neutral N-linked (high mannose and hybrid types) is still difficult, this could be rectified by mimicking lectin carbohydrate-binding domains in enrichment materials. Further, combining HILIC with other techniques, such as reversed-phase C18 chromatography, lectins, or strong anion-exchange methods, can help gather more comprehensive information about glycosylation sites and structures.

The results gathered do not solely rely on having a HILIC sorbent; rather, they also can be affected by the mobile phase, the detection method, and additional LC separation techniques. Zirconia-coated sorbents can enhance selectivity and binding for glycopeptides and phosphopeptides, playing key roles in the primary separation process. That said, overall effectiveness depends on chemical interactions between functional groups on the stationary phase and target compounds. Detection methods, such as MS, can be critical for identifying and quantifying enriched compounds, ensuring high sensitivity and selectivity. When combining HILIC with other separation modes, this further refines results, improving resolution and reducing sample complexity for accurate detection.

These advancements have deepened our understanding of the connections between protein glycosylation and disease. Future research will help add to this effort, but according to the scientists, they should also focus on direct glycoprotein enrichment using biological materials for complete protein information and creating efficient separation methods for accurate glycan quantification.

References

(1) Rafique, S.; Yang, S.; Sajid, M. S.; Faheem, M. A Review of Intact Glycopeptide Enrichment and Glycan Separation Through Hydrophilic Interaction Liquid Chromatography Stationary Phase Materials. J. Chromatogr. A 2024, 1735, 465318. DOI: 10.1016/j.chroma.2024.465318

(2) Glycan. Elsevier B.V. 2013. https://www.sciencedirect.com/topics/nursing-and-health-professions/glycan (accessed 2024-9-16)

(3) Schoberer, J.; Shin, Y-J.; Vavra, U.; et al. Protein Glycosylation in the ER. Methods Mol. Biol. 2018, 1691, 205–222. DOI: 10.1007/978-1-4939-7389-7_16

(4) Schwarz, D. S.; Blower, M. D. The Endoplasmic Reticulum: Structure, Function and Response to Cellular Signaling. Cell Mol. Life Sci. 2016, 73 (1), 79–94. DOI: 10.1007/s00018-015-2052-6

(5) Hydrophilic Interaction Liquid Chromatography. MilliporeSigma 2024. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/analytical-chemistry/small-molecule-hplc/hilic (accessed 2024-9-17)