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

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

Volume: 13 Issue: 1

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

Affiliation:

Keywords: Fetuin, filter aided capture and elution, human IgG, LC-MS/MS, lectin enrichment, N-Glycan analysis, Oglycopeptide.

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.

Cite this article as:

Arul Albert-Baskar, Park Jong-Moon, Lee Hookeun, Han Na-Young and Jeon Jongho, Use of Filter-Aided Capture and Elution Protocol for Concurrent Preparation of N-glycan and O-glycopeptides for LC-MS/MS Analysis, Current Proteomics 2016; 13 (1) . https://dx.doi.org/10.2174/1570164613666160413125041