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A Quantitative Glycomics and Proteomics Combined Purification Strategy
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Simultaneous quantification of N- and O-glycans using a solid-phase method.

Shuang Yang1, Yingwei Hu1, Lori Sokoll1

  • 1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Nature Protocols
|May 19, 2017
PubMed
Summary
This summary is machine-generated.

We developed a Glycoprotein Immobilization for Glycan extraction (GIG) method for simultaneous analysis of N- and O-glycans. This protocol enables quantitative glycomic profiling from a single sample, applicable to high-throughput platforms.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Glycobiology

Background:

  • Glycosylation, the attachment of glycans to proteins, critically influences protein function.
  • N-linked and O-linked glycosylation are major forms, but simultaneous analysis is challenging due to differing release methods.
  • Existing methods struggle to integrate enzymatic and chemical cleavage for comprehensive glycan analysis.

Purpose of the Study:

  • To present a robust protocol for the simultaneous extraction and analysis of both N- and O-glycans.
  • To enable quantitative glycomic profiling from a single biological specimen.
  • To adapt the method for high-throughput automated platforms.

Main Methods:

  • Protein immobilization on an aldehyde-active support via reductive amination.
  • Stabilization of sialic acids using carbodiimide coupling.
  • Enzymatic release of N-glycans (PNGase F) and chemical release of O-glycans (β-elimination with PMP).
  • Mass spectrometry (LC-MS/MS) analysis coupled with the GIG Tool software for glycan identification and QUANTITY tags for N-glycan quantification.

Main Results:

  • The Glycoprotein Immobilization for Glycan extraction (GIG) method allows simultaneous analysis of N- and O-glycans.
  • The protocol provides quantitative data for both glycan types from a single sample.
  • The GIG method is compatible with high-throughput automated systems.

Conclusions:

  • The GIG protocol offers a streamlined approach for comprehensive glycomic analysis.
  • This method overcomes limitations in analyzing diverse glycosylation types simultaneously.
  • The GIG protocol is a valuable tool for advancing glycoproteomics research.