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Related Concept Videos

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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Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry
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Reducing Interferences in Glycosylation Site Mapping.

Lily Birx1, Alex Harvey2, Marla Popov3

  • 1Complex Carbohydrate Research Center (CCRC) and Departments of Biochemistry and Molecular Biology and Chemistry University of Georgia AthensGeorgia30602 USA.

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|February 9, 2023
PubMed
Summary

Identifying N-linked glycosylation sites using peptide-N-glycosidase F (PNGase F) can lead to false positives due to Asn deamidation. This study highlights the issue and offers a strategy to distinguish true glycosylation sites from artifactual deamidation for accurate protein analysis.

Keywords:
GlycansGlycoproteinLiquid chromatography–mass spectrometryglycosylation site mapping

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Area of Science:

  • Biochemistry
  • Proteomics
  • Analytical Chemistry

Background:

  • N-linked glycosylation site mapping is crucial for understanding protein function.
  • Peptide-N-glycosidase F (PNGase F) is commonly used to identify glycosylation sites by converting Asn to Asp.
  • Spontaneous deamidation of Asn residues can interfere with this method, causing false positives.

Purpose of the Study:

  • To alert researchers to potential errors in N-linked glycosylation site identification caused by Asn deamidation.
  • To propose a method for distinguishing true glycosylation sites from artifactual deamidation.

Main Methods:

  • Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze peptide mass changes after PNGase F treatment.
  • Differentiating between native Asp (n-Asp) and iso-Asp (i-Asp) based on chromatographic peak profiles.
  • Analyzing the consensus sequence Asn-X-Ser/Thr for characteristic mass shifts.

Main Results:

  • PNGase F deglycosylation yields a 1-Da mass shift resulting in n-Asp.
  • Chemical deamidation produces both n-Asp and i-Asp, leading to two distinct chromatographic peaks.
  • True N-linked glycosylation sites are identified by a single n-Asp peak, while deamidation sites show dual peaks.

Conclusions:

  • Asn deamidation is a significant source of false positives in PNGase F-based glycosylation site mapping.
  • Distinguishing between n-Asp and i-Asp via LC-MS/MS is essential for accurate site identification.
  • Implementing this strategy enhances the reliability of N-linked glycosylation analysis.