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A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples
Published on: October 1, 2009
Lectin affinity chromatography using porous polymer monolith assisted nanoelectrospray MS/MS
Mohamed Bedair1, Richard D Oleschuk
1Department of Chemistry, Queen's University, Kingston, Ontario, Canada.
The Analyst
|November 25, 2006
Summary
This study introduces an affinity porous polymer monolith (PPM) as a dual-function device for glycan analysis. The PPM acts as a nanoelectrospray emitter and an online affinity capture column, enabling sheathless, zero-dead-volume coupling to mass spectrometry for enhanced glycan preconcentration and detection.
Area of Science:
- Analytical Chemistry
- Materials Science
- Biochemistry
Background:
- Microfluidics coupled with mass spectrometry (MS) offers powerful analytical capabilities.
- Sheathless, zero-dead-volume interfaces are crucial for sensitive and efficient analyte transfer.
- Porous polymer monoliths (PPMs) present tunable properties for various separation and preconcentration applications.
Purpose of the Study:
- To develop and characterize an affinity porous polymer monolith (PPM) for simultaneous nanoelectrospray ionization (ESI) and online affinity-based glycan preconcentration.
- To investigate the influence of PPM pore size on electrospray stability.
- To demonstrate the utility of the affinity PPM for the enrichment and MS detection of specific glycopeptides.
Main Methods:
- Photopolymerization of glycidyl methacrylate/ethylene dimethacrylate to create PPMs with varying pore sizes using different aliphatic alcohol porogens.
- Immobilization of Concanavaline A lectin onto the PPM for affinity capture of high mannose glycans via Schiff base chemistry.
- Characterization of PPM pore size by flow back pressure measurements and electrospray stability by total ion current (TIC).
- Preconcentration and tandem MS analysis of glycopeptides from Ribonuclease B using the affinity PPM sprayer.
Main Results:
- PPM pore size was successfully tuned by varying the alkyl chain length of the porogenic alcohol, with longer chains yielding smaller pores and higher back pressure.
- Smaller PPM pore sizes resulted in enhanced electrospray stability, indicated by a more stable total ion current (TIC).
- The amount of immobilized Concanavaline A varied with the porogenic solvent used during polymerization.
- The affinity PPM effectively preconcentrated glycopeptides from Ribonuclease B, enabling their subsequent detection by tandem MS.
Conclusions:
- Affinity porous polymer monoliths serve as effective, dual-function devices for sheathless, zero-dead-volume coupling of microfluidics to mass spectrometry.
- The developed affinity PPM enables online preconcentration and MS detection of specific glycopeptides, showcasing its potential for glycomic analysis.
- Pore size engineering of PPMs is a critical factor for optimizing electrospray stability in such integrated systems.
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