High sensitivity capillary electrophoresis with fluorescent detection for glycan mapping

Area of Science:

• Analytical Chemistry
• Biochemistry
• Biotechnology

Background:

• Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is crucial for glycan analysis.
• Conventional methods face limitations in detection sensitivity and peak capacity for glucose oligomers and released glycans.
• Existing protocols often rely on polymer-based electrolytes and specific capillary coatings.

Purpose of the Study:

• To develop a novel, polymer-free background electrolyte (BGE) strategy for enhanced CE-LIF analysis of glucose oligomers and glycans.
• To improve detection sensitivity, peak capacity, and the degree of polymerization (DP) visualization in glycan analysis.
• To introduce a new electrokinetic preconcentration strategy for substantial signal enhancement without specialized capillaries.

Main Methods:

• Exploited a polymer-free BGE using triethanolamine/citric acid or triethanolamine/acetic acid at high ionic strengths (up to 200 mM).
• Implemented a novel electrokinetic preconcentration strategy using large volume sample stacking with electroosmotic modulation by varying buffer ionic strength.
• Applied the developed method to analyze N-linked oligosaccharides from a model glycoprotein (Human Immunoglobulin G) and human serum samples.

Main Results:

• Achieved up to a 5-fold improvement in detection sensitivity compared to conventional CE-LIF protocols.
• Enabled visualization of higher degrees of polymerization (DP 18 vs 15) for glucose oligomers.
• Demonstrated a 200-fold signal enhancement using the electrokinetic preconcentration strategy, allowing flexible pH selection for separation.

Conclusions:

• The polymer-free BGE strategy significantly enhances CE-LIF performance for glucose oligomers and glycans.
• The proposed electrokinetic preconcentration method offers substantial signal amplification without requiring neutrally coated capillaries.
• The approach is effective for analyzing complex glycan structures and has potential applications in diagnosing congenital disorders of glycosylation (CDG).