박상혁 (국립창원대학교)

임재민 (국립창원대학교)

박상혁 (국립창원대학교)

권희영 (국립창원대학교 첨단 나노 과학기술 연구원)

우현주 (경남생명항노화 핵심지원센터)

장영훈 (국립창원대학교)

임재민 (국립창원대학교)

Glycosylation is a major post-translational modification (PTM) associated with protein function and various diseases, including cancer. Despite recent advances in glycomics, achieving high quantitative accuracy remains challenging, whereas metabolic stable isotope labeling enables accurate internal standard-based quantification. In this study, a metabolic stable isotope labeling technique for glycans, called MILPIG, was applied to the human lung cancer cell line A549 to optimize labeling conditions and evaluate its applicability in mammalian cells. Stable isotope introduction was carried out via the Hexosamine Biosynthetic Pathway (HBP). A549 cells were cultured in media containing either natural glucose (Light) or ¹³C₁-glucose (Heavy), and isotope incorporation efficiency was monitored under varying culture conditions. The light- and heavy-labeled glycan samples were then spiked at defined ratios and subjected to mass spectrometry. Successful stable isotope incorporation into N-linked glycans was achieved in A549 cells, and optimal metabolic labeling conditions were established. The observed light-to-heavy glycan signal ratios closely matched the expected mixing ratios, demonstrating the feasibility of MILPIG for internal standard-based quantitative glycomics in mammalian cells. These results demonstrate the successful extension of the MILPIG platform to mammalian cells, providing a robust platform for quantitative glycomics and the investigation of disease-associated glycan alterations.

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).