Glycans, Glycosite, and Intact Glycopeptide Analysis of N-Linked Glycoproteins Using Liquid Handling Systems

Area of Science:

• Glycoproteomics
• Mass Spectrometry
• Biochemical Analysis

Background:

• Aberrant glycosylation is linked to disease progression.
• Glycoproteins in biological fluids are key targets for disease monitoring.
• Current glycoproteomic analysis requires efficient sample preparation for large cohorts.

Purpose of the Study:

• To develop a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs).
• To enable rapid and reproducible glycoproteomic analysis for disease monitoring.
• To address limitations in sample preparation for large-scale glycomic studies.

Main Methods:

• Combined hydrophilic and hydrophobic peptide enrichment strategies.
• Utilized a liquid handling platform for high-throughput IGP enrichment in a 96-well format.
• Employed a C18/MAX-Tip workflow for selective IGP enrichment and subsequent analysis of deglycosylated peptides and glycans.

Main Results:

• Developed and optimized a rapid C18/MAX-Tip workflow for IGP enrichment.
• Significantly reduced sample processing time for glycoproteomic analysis.
• Successfully characterized glycans, glycosites, and IGPs from standard and biological samples (urine).

Conclusions:

• The novel method enables high-throughput enrichment of glycan, glycosites, and IGPs.
• This approach facilitates efficient glycoproteomic analysis for disease monitoring.
• The developed workflow is suitable for investigating N-linked glycoproteins in biological samples.