Glycosylation, one of the most common types of post-translational modification (PTM), is
frequently observed in membrane and plasma proteins. Characterization of glycan structures (participating
in glycosylation) using mass spectrometry is important in biopharmaceutical industry. The present
study describes a novel and improved N-glycan enrichment method using a filter aided capture
and elution protocol. The glycopeptides of human IgG digests were selectively captured by binding to
lectins, and the remaining non-glycopeptides were washed off by allowing them to pass through the
membrane. The lectin binding glycopeptides were treated with Peptide -N-Glycosidase F (PNGase F), which cleaves the
bond between arginine and glycan, and the de-glycosylated peptides were selectively obtained by filter-aided capture and
elution method. After eluting the de-glycosylated peptides, the N-glycans and O-glycopeptides attached to the lectins were
released by washing with 80% acetonitrile. The eluted N-glycan moieties and the intact O-glycopeptides were directly injected
to the LC-MS/MS system without further enrichment. We identified 22 N-glycan moieties from a single standard
human IgG protein. This novel protocol allows the enrichment and elution of N-Glycan and intact O-glycopeptides from a
single experimental batch.
Keywords: Fetuin, filter aided capture and elution, human IgG, LC-MS/MS, lectin enrichment, N-Glycan analysis, Oglycopeptide.
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