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Development and Applications of Stable Isotope Labelling Liquid Chromatography Mass Spectrometry for Quantitative Proteomics Open Access


Other title
mass spectrometry
isotope labelling
Type of item
Degree grantor
University of Alberta
Author or creator
Lo, Andy
Supervisor and department
Li, Liang (Chemistry)
Examining committee member and department
Li, Liang (Chemistry)
Weiner, Joel H. (Biochemistry)
Klassen, John S. (Chemistry)
Perreault, Hélène (Chemistry, University of Manitoba)
West, Frederick G. (Chemistry)
Department of Chemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
This thesis describes the characterization, automation, and applications of a stable isotope labelling method for quantitative proteomics by mass spectrometry. Known as 2MEGA, the method uses guanidinylation to convert peptide lysine residues to homoarginine followed by reductive methylation of free peptide N-termini with isotopically encoded formaldehyde. 2MEGA was shown to be applicable to the identification of membrane proteins by increasing the percentage of lysine-containing peptides identified and by the observation of diagnostic a1-ions for >95% of glycine N-terminal peptides and >99% of non-glycine N-terminated peptides. Subsequent work demonstrated that 2MEGA was readily automatable with a commercially available liquid handler. With minor reagent substitutions, the 2MEGA labelling method was used to simultaneously process twelve samples. Over 98% labelling efficiency was observed, with the most common side reaction products being N-terminal guanidinylation (~2%) for glycine and alanine N-terminal peptides. Various front-end protein sample preparation methods were found to be compatible with the procedure. Reciprocal labelling was used to evaluate the internal consistency from quantitative peptide sample comparisons by switching the original isotopic labelling assignment and analyzing the resultant sample mixture. With approximately 60% overlap in peptide identifications, reversal of the isotopic labels was not found significantly affect the observed quantification ratios, as evidenced by the internal consistency in the peptide quantification ratios (an average of 1.29-fold relative difference for the entire E. coli dataset). For over 90% of peptides, the relative error was less than 50%. After discarding 1% of the quantified peptides, approximately 1% of protein matches were lost, but with significant gains in the internal consistency of quantification values. The large-scale quantitative analysis suggested that data processing can greatly influence the overall consistency observed in proteomics experiments. Reciprocal labelling was also applied to the analysis of a human carcinoma cell line deficient in Bax, a key protein in stimuli-induced apoptosis, in which 200 proteins with a significant change abundance difference were identified from Bax-expressing and Bax-deficient samples. Overall, the thesis highlights the potential of the 2MEGA labelling method, its applicability to high throughput MS-based proteomics applications, and suggests an increased role for quantitative mass spectrometry as a routine bioanalytical methodology.
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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