Development of Citric-Acid-Modified Cellulose Monolith for Enriching Glycopeptides

Area of Science:

• Biomaterials Science
• Analytical Chemistry
• Biochemistry

Background:

• Effective enrichment of low-abundance glycopeptides is crucial for mass spectrometry (MS)-based protein glycosylation analysis.
• Current methods often require complex pretreatment steps, impacting efficiency and environmental footprint.

Purpose of the Study:

• To develop an environmentally friendly and efficient medium for glycopeptide enrichment.
• To evaluate the performance of this novel medium for analyzing complex biological samples.

Main Methods:

• Fabrication of a citric-acid-modified cellulose monolith (CCM) using thermally induced phase separation (TIPS).
• Utilizing CCM as a hydrophilic interaction liquid chromatography (HILIC) stationary phase.
• Testing CCM with model proteins (IgG, BSA) and human hepatocellular carcinoma tissue.

Main Results:

• CCM demonstrated a coral-like porous structure and high-density hydrophilic groups.
• Achieved selective enrichment of glycopeptides from IgG even with a 1000:1 ratio of BSA contaminant.
• Identified 35 glycopeptides from IgG with a low detection limit and good reusability.
• Successfully identified 641 unique N-glycosylation sites from human cancer tissue.

Conclusions:

• CCM is a promising, eco-friendly HILIC stationary phase for efficient glycopeptide enrichment.
• This material facilitates improved identification of protein glycosylation in complex biological samples.
• The developed method offers a sustainable approach for glycopeptide analysis in clinical research.