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Related Concept Videos

Protein Glycosylation01:25

Protein Glycosylation

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Glycosylation, the most common post-translational modification for proteins, serves diverse functions. Adding sugars to proteins makes the proteins more resistant to proteolytic digestion. Glycosylated proteins can act as markers and receptors to promote cell-cell adhesion. Additionally, they have many essential quality control functions in the cell, such as correct protein folding and facilitating transport of misfolded proteins to the cytosol, which can be degraded.
Glycosylation occurs in...
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Oligosaccharide Assembly01:24

Oligosaccharide Assembly

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Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
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Proteoglycans01:05

Proteoglycans

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Glycans, a class of complex heterogeneous molecules, can be covalently attached to proteins to form glycosylated proteins that regulate various physiological and pathological processes. Glycosylated proteins or glycoproteins comprise N-linked and O-linked oligosaccharides. O-glycosylation is the most common type of protein glycosylation. Here, glycans attach to the oxygen atom of the hydroxyl groups of Serine or Threonine residues. O-linked glycosylation occurs later in protein processing,...
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Related Experiment Video

Updated: Feb 20, 2026

A Quantitative Glycomics and Proteomics Combined Purification Strategy
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A Method for Mapping Glycosylation Sites in Proteins.

Mark S Han1, John T Simpson2

  • 1Reference Standards Laboratory, The United States Pharmacopeial Convention, Rockville, Maryland 20852, USA; and.

Journal of Biomolecular Techniques : JBT
|October 18, 2017
PubMed
Summary

Quantifying O-linked glycans on glycoproteins is challenging. This study introduces a novel resin-bound protease digestion method for accurate site-specific glycan analysis using mass spectrometry.

Keywords:
LC/MSglycansquantificationsite-specific glycosylation

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Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases
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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Mass Spectrometry

Background:

  • Protein glycosylation analysis, particularly O-linked glycans, is technically challenging.
  • Existing methods like HPLC with fluorescent labeling are not suitable for O-linked glycans.
  • Mass spectrometry (MS) based site-specific glycan analysis is hindered by protease miscleavages.

Purpose of the Study:

  • To develop a new method for site-specific quantification of glycans on glycoproteins.
  • To overcome limitations of existing techniques for O-linked glycan analysis.
  • To enable accurate relative quantification of glycans using mass spectrometry.

Main Methods:

  • Utilized resin-bound, thermally stabilized proteases for native protein digestion.
  • Employed a single-step protocol with resin-bound trypsin on a human monoclonal antibody (mAb).
  • Analyzed resulting glycopeptides using liquid chromatography/mass spectrometry (LC/MS) with hydrophilic interaction chromatography.

Main Results:

  • The novel enzymatic digestion method effectively eliminated miscleavages around glycosylation sites.
  • Site-specific glycopeptides were successfully generated and analyzed from the Fc region of a human mAb.
  • Quantitative analysis showed a high correlation (0.87) between the glycopeptide and released glycan methods.

Conclusions:

  • This resin-bound protease digestion method offers a robust approach for site-specific glycan quantification.
  • The technique overcomes previous limitations in analyzing O-linked glycans.
  • It enables accurate and reliable glycopeptide analysis via LC/MS for glycoprotein characterization.