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Design and optimization of porous polymer enzymatic digestors for proteomics

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Design and optimization of porous polymer enzymatic digestors for proteomics

Lin, Wei (2009) Design and optimization of porous polymer enzymatic digestors for proteomics. Masters thesis, Concordia University.

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Abstract

Effective protein characterization and identification is a demanding and time-consuming operation in proteomics because of long protein purification/separation procedures and even longer enzymatic digestions. In this work, polymer-based monolithic enzyme reactors were fabricated in fused-silica capillaries, and performance was characterized through protein digestion and identification by MALDI-MS and ESI-MS. Trypsin and Staphylococcus aureus V-8 protease (Glu-C) were used to produce three types of reactors: trypsin-based, Glu-C based and tandem trypsin Glu-C. Reactors were prepared by synthesizing a porous methacrylate base monolith followed by photografting with glycidyl methacrylate, and immobilization of the enzyme(s) with carbonyldiimidazole. Protein digestions, performed by perfusing protein solutions through the reactor under pressure, were evaluated based on the peptide map generated when directly coupled to an ESI mass spectrometer. Excellent digestion efficiencies were observed over a flow rate range from 0.2 to 1 oL min -1 , which corresponds to reactor residence times of 1.4 to 0.24 min. As a proof of principle application of a complete proteomics analysis, chromatographic separation of model proteins followed by digestion of specific fractions using these proteolytic enzyme reactors and ESI-MS is demonstrated.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Lin, Wei
Pagination:xii, 87 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry
Date:2009
Thesis Supervisor(s):Skinner, C
ID Code:976464
Deposited By: Concordia University Library
Deposited On:22 Jan 2013 16:26
Last Modified:18 Jan 2018 17:42
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