Use of Filter-Aided Capture and Elution Protocol for Concurrent Preparation of N-glycan and O-glycopeptides for LC-MS/MS Analysis

Author(s): Albert-Baskar Arul , Jong-Moon Park , Hookeun Lee , Na-Young Han , Jongho Jeon .

Journal Name: Current Proteomics

Volume 13 , Issue 1 , 2016

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Abstract:

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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Article Details

VOLUME: 13
ISSUE: 1
Year: 2016
Page: [48 - 54]
Pages: 7
DOI: 10.2174/1570164613666160413125041

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