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A potential pitfall in 18O-based N-linked glycosylation site mapping
Peggi M Angel1, Jae-Min Lim, Lance Wells
1Complex Carbohydrate Research Center and the Departments of Biochemistry and Molecular Biology and Chemistry, University of Georgia, 315 Riverbend Road, Athens, GA 30302-4712, USA.
Rapid Communications in Mass Spectrometry : RCM
|February 7, 2007
Summary
A common method for identifying N-linked glycosylation sites produces high false positives due to active trypsin. This study identifies the cause and offers solutions to improve N-linked glycosylation site identification accuracy.
Area of Science:
- Biochemistry
- Proteomics
- Glycobiology
Background:
- N-linked glycosylation site identification commonly uses tryptic digestion and PNGase F with (18)O labeling.
- A 3 Da mass tag is typically used for detection via liquid chromatography/tandem mass spectrometry (LC/MS/MS).
- A high false positive rate (60%) was observed in identifying glycosylated asparagines not fitting the consensus sequence.
Purpose of the Study:
- To investigate the source of high false positive rates in N-linked glycosylation site identification.
- To identify strategies to overcome the observed false positive phenomenon.
Main Methods:
- Analysis of tryptic digestion and PNGase F digestion in (18)O water.
- Investigation of residual trypsin activity post-proteolysis and cleanup.
- Demonstration of solutions on a model protein.
Main Results:
- Residual trypsin activity led to (18)O incorporation at peptide C-termini, mimicking glycosylated sites.
- This mass shift confused database search algorithms, causing false positives.
- Proposed methods include removing trypsin before deglycosylation, using natural water, or variable modification searches.
Conclusions:
- Residual trypsin activity is a significant source of false positives in N-linked glycosylation site identification.
- Strategies to mitigate this issue include optimizing sample preparation and database search parameters.
- Accurate identification of N-linked glycosylation sites requires addressing this enzymatic artifact.
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