강선영 (국립창원대학교)

임재민 (국립창원대학교)

임재민 (국립창원대학교)

강선영 (국립창원대학교)

배유빈 (국립창원대학교)

O-glycosylation is one of the post-translational modification (PTM) process in which sugar molecules attach to oxygen atoms of the serine/threonine residues of proteins. During glycosylation, glycans attached to affected proteins are modified compared to normal proteins. Therefore, it helps diagnose diseases, so it can be used as a biomarker for diseases such as cancer and Alzheimer's. Glycans are a type of complex carbohydrate composed of glycoprotein components and play an important role in determining the biological activity and function of glycoproteins. To decipher the correlation between prevalence and physiological contribution of glycans, many mass spectrometry and stable isotope labeling-based methods have been developed for relative quantification of glycans. Among them, Metabolic Labeling of Glycans Using Isotopic Glucose for Quantitative Glycomics (MILPIG) is an <i>in vivo</i> labeling method that can produce isotope-labeled isotope glycans using isotope glucose (1,2-¹³C₂ glucose) as a carbon source. In this study, we extend quantitative glycosmics by applying MILPIG in yeast. Yeasts were cultured in medium with normal glucose and in 1,2-¹³C₂ isotope-labeled medium, respectively, and mass spectra were obtained after glycan purification. Unlike conventional MILPIG, which used 1-¹³C₁ glucose to produce a mass difference of 1 Da per unit, 1,2-¹³C₂ glucose results in a mass difference of 2 Da per unit per unit, minimizing the overlap of the spectrum. In addition, a quantitative experiment was conducted by applying a manosidase inhibitor. Manosidase is an enzyme that hydrolyzes mannose, and when a manosidase inhibitor is used, the action of mannosidase is suppressed, and specific bonds such as α -1,2, α -1,3 are not broken, resulting in many long glycans. Representative manosidase inhibitors kifunensine and swainsonine were treated during the culture process, and quantitative information was obtained by comparing the peak area of the changed glycan mass spectrum.