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研究生: 蔡沛倫
Tsai, Pei-Lun
論文名稱: 液相層析質譜技術於醣蛋白定性之分析策略
Strategies for Glycoprotein Characterization using Liquid Chromatography Mass Spectrometry
指導教授: 陳頌方
Chen, Sung-Fang
學位類別: 博士
Doctor
系所名稱: 化學系
Department of Chemistry
論文出版年: 2018
畢業學年度: 107
語文別: 中文
論文頁數: 64
中文關鍵詞: NanoLCtotally porousFused-coremonolithicSDVBmass spectrometrymAbGlycopeptideGlycosylation
英文關鍵詞: NanoLC, totally porous, Fused-core, monolithic, SDVB, mass spectrometry, mAb, Glycopeptide, Glycosylation
DOI URL: http://doi.org/10.6345/DIS.NTNU.DC.001.2019.B05
論文種類: 學術論文
相關次數: 點閱:151下載:2
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  • Nanoflow liquid chromatography coupled with electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS) is a powerful tool in proteomics analysis. The optimum conditions for preparing and the performance of a high efficiency polymeric column was compared with micro particle-filled capillary columns, including a totally porous silica C18 column and a HALO® fused core C18 column. Tryptic peptides were used as model compounds for evaluating the performance of three in-house fabricated columns. After optimization, a monolithic capillary column was prepared by the in-situ polymerization of styrene and divinylbenzene (SDVB) within a 50 μm i.d. fused silica capillary using 1-propanol as the porogen. These continuous unitary porous structures are more robust and efficient compared with bead-based columns. Since meter level SDVB column could substantially reduce peptides co-elution and abate the ion suppression, thus permitting the total ion current signal to be significantly enhanced. For the routine identification of peptides, the performances of these three columns were comparable. For glycopeptides, the monolithic SDVB column gave the highest separation efficiency and a total of 20 N-linked glycopeptides could be identified in a tryptic digest of fetuin and bevacizumab (Avastin). The results indicate that an SDVB column possesses great potential for separating hydrophilic peptides. The novel monolithic media described represents a promising addition to the stationary phase used in capillary columns for proteome research. Another part is about glycopeptide analysis. In the development of biosimilar protein drugs, glycan profiling mapping is critical for providing its biosimilarity. The quantitation and structure information of glycans in antibodies or other glycoprotein drugs for pharmaceutical industries is very important. In this study, a sample preparation method using trypsin digestion with RapiGest SF surfactant (Waters) followed by reverse phase nanoLC-MS/MS was compared to the traditional normal phase HPLC-Fluorsecence with 2-AB (2‑aminobenzamide) labeling and the reduced molecular weight analysis. Glycopeptide-based analysis with fast digestion method provides a novel approach for structural analysis of glycans with relative quantitation. The purpose of this research is to evaluate the optimized method for monoclonal antibody (mAb) digested condition for the glycan type determination and their relative abundance of mAb sample by LC-MS/MS. The results indicate that glycopeptide-based analysis used to provide information of the glycans more convenient on mAb’s characterization. In summary, the optimized preparation of styrene-divinylbenzene copolymer column and glycoprotein digestion reaction optimization combined with nanoLC-MS/MS were developed and investigated, which can be beneficial to protein characterization research.

    Table of Content i List of Tables iii List of Figures iv Abbreviations vi Abstract viii 1. Comparison of Poly(Styrene-Divinylbenzene)-based Monolithic and Bead- based Methodologies Used in NANOFLOW LCMS for Proteomic Studies 1 1.1 Introduction 1 1.2 Fused-core 1 1.3 Monolithic 2 1.4 Materials and methods 3 1.4.1 Reagents and materials 3 1.4.2 Procedure for preparing a monolithic column 4 1.4.3 Protease digestion 4 1.4.4 Liquid chromatography separation and mass spectrometry analysis 5 1.4.5 Data analysis 6 1.4.6 Scanning electron microscopy analysis (SEM) 6 1.5 Results and Discussion 7 1.5.1 Monolithic (PS-DVB) column optimization 7 1.5.2 Characteristics of the three different stationary phases 10 1.5.3 Molecular-weight distribution of the tryptic peptides 11 1.5.4 GRAVY value evaluation 12 1.5.5 Glycopeptide separation comparison 13 1.6 Conclusions 14 2. Optimization of Sample Preparation for Glycan Profiling in a Monoclonal Antibody using Liquid Chromatography Tandem Mass Spectrometry 15 2.1 Introduction 15 2.2 Monoclonal antibody 15 2.3 Mass Spectrometry Analysis Strategy for glycosylation of Mab 16 2.4 Materials and methods 18 2.4.1 Reagents and materials 18 2.4.2 Avastin reduced reaction 19 2.4.3 Digestion steps for Avastin 19 2.4.4 Liquid chromatography separation and mass spectrometry analysis 20 2.4.5 Avastin N-Glycan Release Analysis 21 2.4.6 Data analysis 22 2.5 Results and discussion 23 2.5.1 Trypsin digestion optimization 23 2.5.2 Quantitative evaluation for different glycan analytical methods 24 2.6 Conclusions 27 3. References 28

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