LC-MS Analysis on N glycans in Glycoproteins Based on Transglycosylation Reaction of Endo M N175Q and Acetone precipitated Glycopeptide Enrichment

KeyWord:glycoprotein  N-glycan  enzymatic labeling  LC-MS

Abstract:

      A reliable,efficient and simple method was developed for the determination of N glycans in glycoproteins based on the enzymatic labeling and the acetone precipitated glycopeptides enrichment.Taking sialylglycopeptide(SGP) for instance,a 5 fold volume of acetone was confirmed to be appropriate for the selective precipitation of SGP by comparing the enriching effects of adding volumes of acetone on SGP.To confirm the selectivity of acetone precipitation for N-glycopeptides,tryptic digest from bovine ribonuclease B(RNase B) was treated with acetone,following by chemoenzymatic labeling and a transglycosylation reaction utilizing Endo-M N175Q and PDPZ-Boc-Asn-GlcNAc as acceptor,and the PDPZ Boc Asn GlcNAc N-glycans were obtained.The chromatographic separation was conducted on a YMC C18 column using water containing 10 mmol/L HCOONH4-acetonitrile as mobile phase by gradient elution.The analysis was performed by ESI-MS in positive mode.Five high mannose types of N-glycans in RNase B were detected.Results showed that N-glycosylated peptides could be enriched in the precipitate by adding acetone,and the chemoenzymatic labeling was performed in the presence of the glycopeptides and PDPZ-Boc-Asn-GlcNAc as enzymic catalytic substrates.The method was successful in acquiring the comprehensive MS data for five complex types of glycans in fetuin.Therefore,it is a powerful tool for the determination of N-glycans in glycoproteins.