Sample matrix effects on glycopeptide stability by high performance capillary electrophoresis

Area of Science:

• Biochemistry
• Analytical Chemistry
• Glycoscience

Background:

• Capillary electrophoresis (CE) is crucial for analyzing glycoprotein microheterogeneity.
• Sample matrix selection significantly impacts CE performance and glycopeptide stability.
• Acetic acid is commonly used but can affect glycopeptide integrity.

Purpose of the Study:

• To investigate the impact of acetic acid as a sample matrix on glycopeptide stability and electrophoretic behavior.
• To identify alternative sample matrices that enhance glycopeptide stability and CE performance.
• To optimize conditions for high-resolution analysis of glycoprotein digests.

Main Methods:

• High-performance capillary electrophoresis (CE) coupled with electrospray ionization-mass spectrometry (ESI-MS).
• Analysis of glycoprotein digests using acetic acid and propionic acid as sample matrices.
• Time- and concentration-dependent studies of sialic acid hydrolysis and glycopeptide mobility shifts.

Main Results:

• Acetic acid promotes time- and concentration-dependent hydrolysis of sialic acids from glycopeptides, even at 5°C.
• Hydrolysis in acetic acid leads to changes in electrophoretic mobility and ESI-MS spectra, indicating induced peptide structure.
• Asialoglycopeptides showed stable mobility in acetic acid.
• Propionic acid stabilized electrophoretic mobilities, preventing structural changes observed by ESI-MS within 31 hours.
• Propionic acid resulted in migration time reproducibility of <0.1% RSD, excellent peak shapes, and enhanced glycopeptide resolution.

Conclusions:

• Acetic acid is not ideal for preserving glycopeptide integrity during CE analysis due to sialic acid hydrolysis.
• Propionic acid serves as a superior sample matrix for CE of glycoprotein digests, ensuring stability and improving analytical outcomes.
• The use of propionic acid enhances reproducibility, peak shape, and resolution in glycopeptide analysis.