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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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Glycan Release

It is often difficult to perform detailed structural analysis of the glycan when it is attached to the protein or peptide. And, consequently, such analyses are usually conducted on released glycans. Glycans can be released from glycoproteins either chemically using hydrazine or by p-elimination or enzymatically using a variety of endoglycosidases.

Use of Hydrazine

Although popular in the past, hydrazine use has declined greatly in recent years to be replaced by milder enzymatic methods. However, hydrazine does have the advantage of being nonselective with respect to the glycans that are released, a feature not shared by the majority of enzymes. Anhydrous hydrazine is a reagent that cleaves amide bonds and, thus, causes complete disruption of the protein chain. It can be used to release both N- [73-75] and O-linked [74] glycans. O-linked glycans are specifically released at 60 °C, whereas 95 °C is needed to release the N-linked sugars. A major advantage of the method is that the sugars are released with an intact reducing terminus that can subsequently be used to label the glycan with various tags such as the fluorescent tags used for analysis by high-performance liquid chromatography (HPLC). On the other hand, because hydrazine cleaves amide bonds, it removed the acetyl groups from M-acetylamino sugars requiring a re-N-acetylation step in the release procedure. A recent method employs a carbon column for simultaneous reacetylation and hydrazine removal [76]. In addition, formation of artifacts can reduce the yield of glycans. Thus, it has been calculated [77] that as much as 25% of the total glycans are modified at the reducing terminus and that these compounds can never be converted into the parent sugar. Another disadvantage of hydrazine release is that the protein is completely destroyed, resulting in loss of all information regarding glycan linkage position.

 
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