Thermo Scientific Guide to Glycan Analysis Glycosylation is one of the most important post-translational modifications of eukaryotic cell proteins. Glycan-modified proteins are involved in a wide range of biological and physiological processes including recognition and regulatory functions, cellular communication, gene expression, cellular immunity, growth, and development.
Glycan functions are often dependent on the structure of the oligosaccharide. Oligosaccharides are covalently attached to proteins primarily through two structural motifs: attached to the amide group of an asparagine, referred to as “N-linked glycans,” or attached to the hydroxyl group on serine or threonine, referred to as “O-linked glycans”. Both types of glycans are investigated as biomarkers, in order to understand changes related to complex organelle development, and as part of therapeutic protein drug development with strong indication that efficacy is affected by glycosylation.1 Agencies such as the United States Food and Drug Administration (U.S. FDA) and the European Medicines Agency have published documents recommending that biopharmaceutical manufacturers demonstrate satisfactory programs for understanding, measuring, and controlling glycosylation in glycoprotein-based drugs, with recent updated draft guidance from the U.S. FDA for characterization of biosimilar protein therapies.2 This guidance suggests that the oligosaccharide content of glycoprotein products should be understood and monitored to ensure product consistency. Many analytical approaches have been employed in the area of glycan and glycoproteomics research, which can be generally summarized under the following categories:
• Monosaccharide analysis
• Glycan analysis
• Glycosylation site profiling
• Intact glycoprotein profiling
These analyses are performed to determine the identity and quantity of the carbohydrates present, the post-translational modification site, and the multiple glycoforms of a protein that may be present. Choosing the appropriate analysis technique depends on experimental demands and instrumental capabilities. This handbook is intended to assist the decision-making process.