The LudgerLiberate Hydrazinolysis Kit contains reagents for the release of N and O-linked glycans from glycoprotein biopharmaceuticals. Released glycans have free reducing terminii to allow fluorescent tagging by reductive amination. The release conditions can be optimized for release of N-glycans, O-glycans or both N- and O-glycans. An alternative method for the release of O-glycans is the LudgerLiberate ORELA Kit (LL-ORELA-A2).
The kit contains reagents and materials for up to 12 glycoprotein samples analysed in parallel or two sets of 6. Up to 1 mg of glycoprotein can be deglycosylated per sample.
Store refrigerated at 4 – 10˚C in the dark. If you have limited cold storage space then store just the CEX cartridges at 4˚C and the rest of the kit at room temperature. Protect from sources of heat, light, and moisture. When stored correctly, the reagents should be stable for at least 18 months from date of manufacture.
The product should be shipped at ambient temperature. As it contains small amounts of anhydrous hydrazine the kit must be packaged and shipped as dangerous goods
The Hydrazinolysis Reaction
1. Liberation of the glycan as the hydrazide derivative.
Anhydrous hydrazine reacts at the link between the glycan and peptide backbone and the acetamido groups of monosaccharide residues such as GlcNAc. This results in liberation of the glycan as the de-N-acetylated hydrazone derivative which then converts to the hydrazide form.
Free amino groups on the hydrazide are N-acetylated to form the β-acetohydrazide derivatives. This repairs the de-N-acetylated monosaccharide residues and caps the hydrazide moiety of the monosaccharide residue formerly linked to the peptide backbone.
3. Hydrolysis to form the free glycan.
The acid-labile β-acetohydrazide derivative is hydrolysed to produce the free glycan.
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Kozak RP, Royle L, Gardner RA, Fernandes DL, Wuhrer M. Suppression of peeling during the release of O-glycans by hydrazinolysis. Anal Biochem. 2012 Apr 1;423(1):119-28.
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Turyan I, Hronowski X, Sosic Z, Lyubarskaya Y. Comparison of two approaches for quantitative O-linked glycan analysis used in characterization of recombinant proteins. Anal Biochem. Feb 1;446:28-36 (2014).