배유빈 (국립창원대학교)

임재민 (국립창원대학교)

배유빈 (국립창원대학교)

권희영 (국립창원대학교 첨단나노과학기술연구소)

우현주 (경남생명항노화 핵심지원센터)

장영훈 (국립창원대학교)

임재민 (국립창원대학교)

Glycosylation, a post-translational modification (PTM), involves the covalent attachment of glycans to proteins or lipids. This process plays a critical role in regulating essential biological functions. O-glycosylation, which involves the attachment of glycans to the hydroxyl groups on serine or threonine residues, is essential for modulating protein activity and intracellular signaling pathways. However, the inherent structural complexity of O-glycans has presented analytical challenges in the past. To address these challenges, various mass spectrometry-based relative quantification methods combined with stable isotope labeling have been developed.

In this study, we developed an O-glycan quantification platform using the Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose (MILPIG) technique in the A549 human lung cancer cell line. This technique supplies isotope-labeled glucose (1-¹³C₁ or 1,2-¹³C₂ glucose) during the cell culture stage to naturally incorporate the isotope within the glycosylation pathway through intracellular hexosamine biosynthetic pathway (HBP). This strategy induces a mass change of 1 Da or 2 Da per unit, improving spectral resolution and analytical efficiency. The MILPIG-based analytical platform established in this study effectively addresses the structural complexity of O-glycans and provides a robust foundation for obtaining highly reliable quantitative data.

Acknowledgements: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2024-00463300), and by the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (2023R1A6C101B022).