PhD Scientific Days 2026

Pharmaceutical Sciences and Health Technologies 1.

Enrichment of Glycosaminoglycan-Linker Glycopeptides Using a Combination of Size-Exclusion Chromatography and High-pH Reversed-Phase Fractionation

Name of the presenter

Dessidianti, Rachma

Institute/workplace of the presenter

1. MTA-HUN-REN TTK Lendület (Momentum) Glycan Biomarker Research Group, HUN-REN Research Centre for Natural Sciences, Budapest, Hungary; 2. Semmelweis University Doctoral College, Budapest, Hungary

Authors

Rachma Dessidianti¹, Dávid Virág², Gábor Kecskeméti³, Zoltán Szabó³, Lilla Turiák²
1: 1. MTA-HUN-REN TTK Lendület (Momentum) Glycan Biomarker Research Group, HUN-REN Research Centre for Natural Sciences, Budapest, Hungary; 2. Semmelweis University Doctoral College, Budapest, Hungary
2: MTA-HUN-REN TTK Lendület (Momentum) Glycan Biomarker Research Group, HUN-REN Research Centre for Natural Sciences, Budapest, Hungary
3: Department of Medical Chemistry, Albert Szent-Györgyi Medical School, University of Szeged, Szeged, Hungary

Text of the abstract

Proteoglycans (PG) are a specific type of glycoproteins in which one or more linear glycosaminoglycan (GAG) chain is covalently attached to serine (Ser) residues via characteristic tetrasaccharide linker (GlcA-Gal-Gal-Xyl-O-Ser). The investigation of site-specific PG glycosylation is essential for deeper understanding of glycoprotein activities in physiological contexts. The structural complexity, low abundance, and diversity of GAG side chains require the development of enrichment methods that selectively identify these glycopeptide subsets, which are essential for glycoproteomic studies.
The objective of this research is to develop an effective enrichment method for GAG-linker glycopeptides using decorin as a model PG by integrating size-exclusion chromatography (SEC) with high-pH Reversed-Phase fractionation. A comparative analysis of this combined method towards previous GAG-linker glycopeptide enrichment methodologies is provided.
Decorin containing a single chondroitin sulfate (CS) GAG chain was subjected to digestion with PNGase F and Lys-C-trypsin to cleave N-glycans and proteins, respectively. The enrichment of GAG glycopeptides and desalting steps were performed using custom-packed SEC cartridges loaded with Sephadex G-75 gel. The CS chains were then digested with Chondroitinase ABC. Subsequently, high-pH reversed-phase fractionation was conducted using 8-stepwise elution solutions composed of acetonitrile and 0.1% triethylamine in different ratios. Measurements were performed on Waters nanoAcquity nanoUHPLC coupled to Thermo Fisher Exploris 240 Orbitrap MS.
According to the extracted ion chromatograms, 21 diagnostic oxonium ions were identified. Fractions 3-7 indicated the presence of CS-linker glycopeptides and fraction 4 contained the highest number of CS-linker glycopeptides. The structures of 22 CS-linker glycopeptides were identified exceeding previously reported results, despite using only one hundredth of the sample amount employed in those studies.
The utilization of SEC in conjunction with high-pH RP fractionation is a novel strategy for the enrichment of CS-linker glycopeptides. The approach is sensitive enough to detect amounts relevant for clinical applications. These findings indicate its prospective implementation in clinical samples for biomarker detection.
Funding: Lendület (Momentum) Program of the Hungarian Academy of Sciences.