Strategies for Glycoprotein Characterization using Liquid Chromatography Mass Spectrometry

• Main Authors: Tsai, Pei-Lun, 蔡沛倫
• Other Authors: Chen, Sung-Fang
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/srf7a2

博士 === 國立臺灣師範大學 === 化學系 === 107 === 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.