A Systematic Optimization Analysis of N-Linked Glycans on Porous Graphitic Carbon (PGC) at Different Column Temperatures

Abstract

Protein glycosylation is a post-translational modification where glycans are covalently attached to proteins. N-linked glycosylation is a type of protein glycosylation which involves the covalent attachment of an oligosaccharide to an asparagine residue within a polypeptide chain. Glycans are involved in a number of biological processes such as cell-cell interactions, cell adhesion, immune recognition, and protein folding and function. However, aberrant glycosylation is mostly associated with many types of diseases such as cancer, autoimmune disorders and many other diseases. Therefore, understanding the variations in glycomics profiles on protein has become very crucial for molecular testing. Despite the advance in LC-MS/MS for structural diverse N-glycan analysis, the separation and analysis of N-linked glycans are analytically challenging due to the molecule large number of possible isomeric conformations, difficult to ionize and contain a variety of complex composition, branching and linkage isomers. Even though, many reversed PGC phases have been used for the separation of N-glycans, especially in the context of LC-MS techniques but is still difficult to implement in a robust and reproducible manner. Herein, systematic optimization analysis of N-linked glycans in porous graphitic carbon (PGC) at different column temperatures was investigated. Underivatized tri-sialylated species and bi-sialylated species N-glycans derived from bovine fetuin and human transferrin respectively were utilized to optimize at different temperature. The study suggests that by optimizing at higher temperature, PGC columns can significantly enhance the resolution of complex glycan mixtures, offering powerful tools for in-depth glycomics optimization analysis.