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Rhinogen ^® Protein Deglycosylation Kit Ⅲ (for N-Linked & Complex O- Linked Glycans) contains the enzymes and reagents required to remove all N-linked and some complex O -linked glycans. The enzyme reagents are a mixture of five glycosidases, including PNGase F, O -Glycosidase, α2-3.6.8.9Neuraminidase, β1-4 Galactosidase and β-N- Acetylhexosaminisase . All enzymes and reagents contained in the kit are compatible with downstream HPLC and MS. PNGase F is an endoglycosidase that specifically removes N-linked glycans from glycoproteins. It can release most N-linked glycans (except the innermost GlcNac with α-1,3-fucose residues, which are commonly found in plant and insect glycoproteins) intact from the protein molecules to which they are attached. It can cut between the innermost N-acetylglucosamine ( GlcNAc ) and asparagine residues ( Asn ) of the high mannose, hybrid and complex oligosaccharide parts of N-linked glycopeptides or glycoproteins to release complete oligosaccharide chains, as shown in Figure 1.

Figure 1. PNGase F can cleave and release almost all types of N- linked glycans from glycoproteins or glycopeptides.

However, for O -linked glycans, due to their complex and diverse structures and larger number, a series of glycosidases need to be used in combination to remove modified monosaccharides from the ends of the sugar chains one by one. α2-3.6.8.9 Neuraminidase releases sialic acid residues at all straight or branched non-reducing ends, β1-4 Galactosidase hydrolyzes β1-4 linked galactose residues on the oligosaccharide chains, and β-N- Acetylhexosaminisase catalyzes the hydrolysis of β-DN-acetylgalactosamine and glucosamine residues at the ends of the glycans until the O -linked glycans are trimmed into Core 1 (Gal-GalNAc- O -S/T) or/and Core 3 ( GlcNAc -GalNAc- O -S/T) structures. Only then can O -Glycosidase cleave and release Gal-GalNAc or/and GlcNAc -GalNAc core disaccharide structures, as shown in Figure 2. This combination of five enzymes cannot enzymatically release all O -linked glycans, such as the rare α-linked galactose, α-linked fucose, N-acetylglucosamine directly O -linked to proteins (found on nuclear proteins), N-acetylgalactosamine directly α-linked to proteins (found in mucins), fucose and mannose directly O -linked to proteins, etc.