Glycoproteomic analysis of therapeutic glycoproteins with sialylated N-glycan linkage isomersusing LC-MS/MS

목차 (Table of Contents)

• Introduction 1
• Chapter Ⅰ: Identification, distinguishment and quantification of N-glycans in glycoengineered CTLA4-Ig by N-glycan analysis 10
• 1.1. Materials and methods 11
• 1.1.1. Expression and purification of CTLA4-Igs 11
• 1.1.2. Preparation of N-glycans 11
• 1.1.3. N-glycan analysis using LC-ESI-HCD-MS/MS 12
• 1.1.4. Structural analysis of N-glycans 13
• 1.1.5. Identification of sialylated N-glycan linkage isomers 13
• 1.1.6. Preparation of N-glycans treated by α2,3 neuraminidase 14
• 1.1.7. Quantitative analysis of N-glycans 14
• 1.2. Results and discussion 15
• 1.2.1. N-glycan analysis using LC-ESI-HCD-MS/MS 15
• 1.2.2. Determination of sialylated N-glycan linkage isomers in MS/MS spectrum 17
• 1.2.3. Determination of sialylated N-glycan linkage isomers by retention time 20
• 1.2.4. Ln/Nn values by intensity of fragment ions 23
• 1.2.5. Scatter plot of Ln/Nn value versus RT 27
• 1.2.6. Summary of N-glycan structures and quantities 29
• 1.2.7. Proportion of sialylated N-glycan linkage isomers 33
• 1.3. Conclusion 51
• Chapter Ⅱ: A strategy to distinguish sialylated N-glycan linkage isomers in N-glycopeptide analysis by fragmentation and multiple linear regression analysis 53
• 2.1. Materials and methods 54
• 2.1.1. Preparation of N-glycopeptides from CTLA4-Ig 54
• 2.1.2. Enrichment of N-glycopeptides 55
• 2.1.3. N-glycopeptide analysis using nano-LC-HCD-MS/MS 55
• 2.1.4. Structural analysis of N-glycopeptides 56
• 2.1.5. Data and statistical analysis 57
• 2.1.6. Nomenclature of N-glycan 58
• 2.2. Results and discussion 58
• 2.2.1. N-glycopeptide preparation 58
• 2.2.2. N-glycopeptide analysis using nano-LC-HCD-MS/MS 59
• 2.2.3. Distribution of N-glycopeptides 63
• 2.2.4. Sialylated N-glycan linkage isomers in N-glycopeptides 64
• 2.2.5. Normalization of fragment ion ratio 67
• 2.2.6. Summary with scatter plot 70
• 2.2.7. Summary with schematic representation 73
• 2.3. Conclusion 93
• References 94
• 국문초록 108