Quantitative Glycomics of O-linked Glycans in Yeast Using Metabolic Isotope Labeling = 대사적 동위원소 표지에 의한 효모 O-연결 글리칸의 정량 글리코믹스

다국어 초록 (Multilingual Abstract)

O-glycosylation in S.cerevisiae is a major post-translational modification in which mannose residues are attached to Ser or Thr residues of proteins, influencing their stability, localization, and function. To investigate glycan structural dynamics, mass spectrometry-based quantitative methods combined with stable isotope labeling have been widely developed. Among them, the MILPIG (Metabolic Isotope Labeling of Glycans Using Isotopic Glucose) strategy enables efficient in vivo labeling of yeast glycans using 1,2-13C2 glucose.
In this study, we applied MILPIG to establish a quantitative O-glycomics workflow in S.cerevisiae. Yeast cultured in unlabeled or 1,2-13C2 glucose incorporated isotopic signatures into O-linked glycans, producing a 2 Da mass shift per mannose unit and improving spectral resolution compared to conventional 1 Da labeling. Relative quantification was achieved by comparing mass spectral peak areas between labeled and unlabeled samples. Using this approach, we observed O-glycosylation changes induced by various conditions, including Kifunensine and Swainsonine treatment, supplementation with mannose analog, and pH alterations.

목차 (Table of Contents)

• LIST OF TABLES
• LIST OF FIGURES
• 1. INTRODUCTION
• 1.1. Protein and PTM
• 1.2. Glycosylation and Glycan
• 1.2.1. Glycosyltaion
• 1.2.2. Glycans
• 1.2.3. Medical Applicants of Glycans
• 1.3. Glycan Analysis by Spectrometry
• 1.3.1. Advantages of Mass Spectrometry
• 1.3.2. Isotope Labeling Methods for Quantitative Glycan Analysis
• 1.4. Glycan of Yeast
• 1.5. Research Objective: Development of Quantitative Methods for O-linked Glycans
• 2. EXPERIMENT
• 2.1. Materials and Chemicals
• 2.2. Sample Harvesting and Glycan Purification
• 2.3. Yeast Culturing
• 2.3.1. Preparation of Media for Yeast Culturing
• 2.3.2. Initial Yeast Culturing and Streaking
• 2.3.3. Yeast Culturing
• 2.3.4. Yeast Culturing under Various Conditions
• 2.4. Yeast Harvesting and Protein Purification
• 2.5. O-linked Glycan Isolation
• 2.6. Permethylated Glycan
• 2.7. Glycan Analysis by Mass Spectrometry
• 2.8. Data Analysis
• 3. RESULTS AND DISCUSSTION
• 3.1. Full MS Spectrum of O-linked Glycans in Yeast
• 3.2. Full MS Spectrum of 1-13C1 labeled O-linked Glycans in Yeast
• 3.3. Full MS Spectrum of 1,2-13C2 labeled O-linked Glycans in Yeast
• 3.4. Individual Spectra in Yeast and Peak Overlap with 1-13C1 Glucose
• 3.5. Resolution of Peak Overlap with 1,2-13C2 Glucose
• 3.6. Quantification of O-linked Glycans with 1,2-13C2 Glucose
• 3.7. Establishing Various Conditions to Ensure Reliable Quantification
• 3.7.1. Quantification of Yeast O-linked Glycans with Mannosidase Inhibitors
• 3.7.2. Quantification of Yeast O-linked Glycans with Mannose Analogs
• 3.7.3. Quantification of Yeast O-linked Glycans under pH Variations
• 4. CONCLUSION
• LITERATURE CITED
• ABSTRACT