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Permanent link (DOI): https://doi.org/10.7939/R3QN5ZJ50

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Development and Applications of Mass Spectrometric Methods for Proteome Analysis and Protein Sequence Characterization Open Access

Descriptions

Other title
Subject/Keyword
microwave-assisted acid hydrolysis
proteomics
mass spectrometry
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Chen, Lu
Supervisor and department
Li, Liang (Chemistry)
Examining committee member and department
Clive, Derrick (Chemistry)
Schriemer, David (Chemistry)
Lucy, Charles A. (Chemistry)
Harynuk, James (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2013-07-11T14:02:17Z
Graduation date
2013-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Mass spectrometry (MS) is widely used in proteomic work for protein identification and characterization. A key to the success of MS for protein analysis is related to sample preparation. Current sample preparation methods do not meet all the needs of protein or proteome analysis. My thesis work is devoted to the development and application of new or improved protein sample preparation methods for MS analysis. These methods are focused on three aspects: microbore LC-UV for quantification of peptides generated from the proteins extracted from a small number of cells, 2-MEGA (N-terminal dimethylation after lysine guanidination) isotope labeling for proteome quantification, and microwave-assisted acid hydrolysis (MAAH) for protein sequence analysis. It is shown that microbore LC-UV is a robust technique for quantifying peptides down to about 40 ng, as well as for removing salts for LC-MS/MS analysis of peptides and this method can be used to gauge the sample integrity during the proteomic sample preparation in dealing with small number of cells. An automated 2-MEGA labeling method utilizing a commercial liquid handler was developed to minimize variability from sample handling during the labeling reaction for high throughput applications. To find biomarkers of breast cancer, the 2-MEGA labeling method was applied to the breast cancer tissues for relative proteomic comparison with the normal breast tissues. 119 proteins were differentially expressed in all three tumor samples and some of these proteins can potentially be verified and validated as biomarkers. To provide a detailed characterization of a protein of interest including amino acid substitution and modifications, MAAH MS was further developed to improve its performance and applicability. Hydrolysis performed in an optimized procedure using a commercial microwave oven was found to be comparable with that in a household microwave oven. A method of characterizing terminal peptides of a protein based on HCl MAAH of proteins, LC fractionation of the hydrolysates and LC-ESI MS/MS analysis of the low molecular weight peptides, was developed. In addition, proteins separated by gel can be hydrolyzed by MAAH to analyze their protein sequences. Both electroelution of proteins from a gel and in-gel MAAH were studied for analyzing gel-separated proteins.
Language
English
DOI
doi:10.7939/R3QN5ZJ50
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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