On-line Coupling of the UV flowcell with Liquid Chromatography-Electrospray Ionization Mass Spectrometry for Characterizing Disulfide Linkages of Proteins

  • 魏 士堯

Student thesis: Doctoral Thesis

Abstract

Depending on the ACS paper “Acetone/Isopropanol Photoinitiating System Enables Tunable Disulfide Reduction and Disulfide Mapping via Tandem Mass Spectrometry’’ Alcohol with 1% acetone under UV 254 nm light can create radicals which can reduce protein disulfide bonds By adjusting UV exposure time the system can control the disulfide bonds reducing level furthermore characterizing disulfide linkages In this paper We combined this UV reaction system with liquid chromatography to make it suitable for complex proteins or peptide samples Or it can separate different reducing level proteins to make disulfide mapping easier We chose water and methanol as the LC mobile phase By changing the ratio of water to methanol and controlling the flow rate we can make sure the reaction efficiency is enough And we used the UV flowcell to control the parameter accurately There were two sites to install UV flowcell One site was after C4 column which could reduce proteins or peptides disulfide bonds after separation This meant we could run a complex sample on the UV flowcell system The other site was before C4 column which could separate different disulfide bond reducing level proteins after the UV reaction By CID MS2 of those different reducing level proteins we could gain the protein disulfide bond information We chose insulin as our model protein After UV reaction the C4 column separated insulin insulin A chain and insulin B chain And then we could get insulin disulfide bond information via CID-MS2 of insulin and insulin A chain We also tried to inject two proteins one time to prove that the UV reaction efficiency was still good under multiple protein samples
Date of Award2019
Original languageEnglish
SupervisorShu-Hui Chen (Supervisor)

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