Glycosylation is one of the most common and functionally important post-translational modifications (PTM) that proteins experience. Each protein molecule with the same sequence of amino acids may be glycosylated in a different location or a different way. The most common glycoforms are well understood and are referred to by the number of terminal galactose residues and presence of fucose – (e.g. G2F is a Glycoform with 2 terminal galactose residues, and with a Fucosylated biantennary complex). The relative abundance of G0, G0F, G1, G1F, G2, G2F, etc. in a sample is referred to as the glycan profile. To determine the glycan profile and glycan sites, two experiments are performed in sequence:
- To determine the location of the glycosylation sites, we digest the protein resulting in glycopeptides and unmodified peptides. Mapping the modified and unmodified peptides back to the amino acid sequence reveals the glycan sites.
- Measure the intact mass of each glycoprotein using liquid chromatography coupled with mass spectrometry to understand their identity and to record the peak area for each glycoform. For antibodies, we can digest with specific enzymes to analyze the Fc and Fab regions.