PhD Scientific Days 2025

Pharmaceutical Sciences and Health Technologies I.

Miniaturized Size Exclusion Columns as an Enrichment Strategy to Improve HPLC-MS-Based Proteoglycan Analysis

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 School, 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 School, Budapest, Hungary
2: 1. MTA-HUN-REN TTK Lendület (Momentum) Glycan Biomarker Research Group, HUN-REN Research Centre for Natural Sciences, Budapest, Hungary
3: 3. Department of Medical Chemistry, Albert Szent-Györgyi Medical School, University of Szeged, Szeged, Hungary

Text of the abstract

Introduction: Proteoglycans (PGs), a subset of glycoproteins, are composed of a core protein which contains covalently linked glycosaminoglycan (GAG) chains attached via a tetrasaccharide linker region. Glycosylation is a crucial post-translational modification, and a comprehensive study of site-specific PG glycosylation is needed to comprehend glycoprotein functions in both normal and pathological processes. The structural complexity, low abundance in biological tissues, and heterogeneity of the GAG side chains require the development of enrichment methods to selectively capture these subsets of glycopeptides, which is essential for glycoproteomic research.
Aims: Our objective was to develop a workflow for the effective enrichment of GAG-linker glycopeptides found in PG decorin by developing miniaturized, custom-packed size exclusion chromatography (SEC) spin columns, and to compare the approach to the available ones.
Methods: PG decorin was digested using PNGase F and Lys-C-trypsin to cleave N-glycans and proteins, respectively. Enrichment of GAG-linker glycopeptides was accomplished with Sephadex G25, G50, G75, and G100 SEC spin columns, followed by Chondroitinase ABC digestion of the chondroitin sulfate (CS) chains. Desalting of the GAG-linker glycopeptides was achieved using C18 spin tips. Measurements were performed on a Waters nanoAcquity nanoUHPLC coupled to a Thermo Fisher Exploris 240 Orbitrap mass spectrometer.
Results: Based on the extracted ion chromatograms, the presence of 5 oxonium ions, [HexNAc−2H2O]+ m/z=168.066, [HexNAc−H2O]+ m/z=186.076, [HexNAc]+ m/z=204.087, [ΔHexAHexNAc]+ m/z=362.108, and [HexAHexNAc]+ m/z =380.119, could be identified indicating that the fraction contained CS-linker glycopeptides. The glycopeptide linker sequence, identified as DEASGIGPEEHFPEVPEIEPMGPVCPFR, along with the seven precise structures of the CS-linker glycopeptides, were determined through database search, demonstrating favorable results with the use of SEC spin columns, especially G75.
Conclusion: The utilization of the newly developed, custom-packed mini size exclusion columns showed superior results for the enrichment of CS-linker glycopeptides compared to existing methods. These findings support their application to clinical samples.
Funding: The project was supported by the Lendület (Momentum) Program of the Hungarian Academy of Sciences.