Glycosylation is an important and common post translational modification in proteins. Glycoproteins are proteins that carry one of more glycans covalently attached to a polypeptide backbone, usually via N- or O-linkages. One of the most abundant glycoproteins in human serum is Immunoglobulin G (IgG), a type of antibody. IgG is composed of four subunits, two identical light chains and two identical heavy chains, forming a structure with twofold symmetry. Both heavy chains have a single N-linked glycosylation site.
Due to IgGs crucial role in the human immune response and their ability to be recombinantly produced for use as therapeutics, IgG characterization is of great interest to the biopharmaceutical field. The glycosylation of IgG is shown to affect anti-inflammatory response and effector function activity, including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Various methods have been developed to characterize IgG glycosylation; with liquid chromatography interfaced to mass spectrometry (LC-MS) being commonly used. Hydrophilic Interaction Chromatography (HILIC) has been shown to be advantageous in the separation of glycopeptides due to the hydrophilicity of the modification.
Here, HILIC chromatography has been shown to separate O-mannose glycopeptides and isomers. This work shows the ability of the HILIC column to separate these glycopeptides and unglycosylated species in a predictable manner, which allows for easier identification, characterization, and quantification. In addition, a HILIC-LC-MS method for reducing interferences for glycosylation site mapping is also proposed. As well as answering the age-old question of, “Do I need to trypsin digest?” before releasing IgG glycans with PNGase F