PhD Scientific Days 2024

Poster Session R - Pharmaceutical Sciences and Health Technologies 2.

Identification of Glycosaminoglycan-Linker Glycopeptides in Proteoglycan Decorin Using HPLC-MS

Text of the abstract

Introduction: Proteoglycans are generally composed of a core protein containing one or more covalently linked glycosaminoglycan (GAG) chains via a tetrasaccharide unit called the linker region. Proteoglycans play roles in normal and cancer cell physiology, with dysregulated expression contributing to cancer metastasis. The field of glycoproteomics focuses on the study of glycoproteins which also involves the identification and characterization of glycosaminoglycan-linker glycopeptides. In order to have a better understanding of the roles of glycoproteins in health and disease, it is essential to have a profound comprehension of the proteoglycan site-specific glycosylation alterations that occur throughout both normal and pathological processes.
Aims: Our aim is to develop a workflow to efficiently enrich and identify the glycosaminoglycan-linker glycopeptides that are present in decorin, a small leucine-rich proteoglycan, and to apply the workflow to more complex biological samples.
Methods: Decorin was sequentially digested using Lys-C/trypsin and chondroitinase ABC enzymes. Enrichment of glycosaminoglycan-linker glycopeptides was accomplished with the use of 10 kDa centrifugal filter membranes. Liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was utilized to identify the glycosaminoglycan-linker glycopeptides.
Results: Based on the extracted ion chromatograms, the presence of three diagnostic oxonium ions, [ΔHexAGalNAc]+ (m/z = 362.11), [GlcAGalNAc]+ (m/z = 380.12), and [GalNAc]+ (m/z = 204.09), could be identified indicating that the fraction contained chondroitin sulfate-linker glycopeptides. The exact structure of the glycopeptides could not be identified using Byonic software as tandem mass spectrometry conditions need further optimization.
Conclusions: The utilization of Lys-C/trypsin and chondroitinase ABC enzymes in conjunction with a 10 kDa centrifugal filter membrane show promising results for the enrichment of chondroitin sulfate-linker glycopeptides.
Further Studies: A comparative analysis will be conducted using different enrichment methods for glycosaminoglycan-linker glycopeptides identification using decorin standard.
Acknowledgments: Support of the Lendület (Momentum) Program of the Hungarian Academy of Sciences is acknowledged. The Authors are grateful to Markus Ralser for donating the nanoAcquity HPLC to the group.