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Glycopeptide Capture for Cell Surface Proteomics
Published on: May 9, 2014
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Optimized Glycopeptide Enrichment Method-It Is All About the Sauce
David Morgenstern1, Hila Wolf-Levy1, Nili Tickotsky-Moskovitz2
1The de Botton Institute for Protein Profiling, Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot 7610001, Israel.
Analytical Chemistry
|June 28, 2022
Summary
Optimizing glycopeptide enrichment using boronic acid requires careful buffer selection. An amine-less buffer significantly enhances glycoproteomic coverage in biological samples, improving glycopeptide quantification.
Area of Science:
- Biochemistry
- Proteomics
- Glycoscience
Background:
- Protein glycosylation is a critical posttranslational modification involved in numerous biological processes and diseases.
- Analyzing the diverse array of glycopeptides presents significant challenges due to the vast number of possible glycan-peptide combinations.
- Existing boronic acid-based enrichment methods for glycopeptides have limitations in achieving comprehensive coverage.
Purpose of the Study:
- To investigate the impact of buffer composition on the efficiency of boronic acid-based glycopeptide enrichment.
- To identify optimal conditions for maximizing glycoproteomic coverage in complex biological samples.
- To improve the enrichment and analysis of N-linked glycopeptides.
Main Methods:
- Utilized boronic acid affinity chromatography for glycopeptide enrichment.
- Systematically evaluated different buffer systems, focusing on the presence or absence of amines.
- Analyzed glycopeptide enrichment efficiency in human plasma and brain tissue specimens using mass spectrometry.
Main Results:
- Demonstrated that amine-less buffer systems significantly outperform traditional buffers for glycopeptide enrichment.
- Achieved over a 10-fold improvement in total quantified glycopeptides compared to standard methods.
- Successfully identified 1598 N-linked glycopeptides in brain tissue and 737 in non-depleted plasma.
Conclusions:
- Buffer choice, specifically the elimination of amines, is a critical factor for successful boronic acid-based glycopeptide enrichment.
- Amine-less buffers enhance glycoproteomic coverage by preventing competition with glycans for boronic acid binding sites.
- This optimized method provides a substantial improvement for glycoproteomic analysis in clinical and research settings.
