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Quantitative Label-Free Comparative Proteomic Analyses of Eukaryotic Tissues via Mass Spectrometry
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- Author / Creator
- Kramer, David A
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In recent years, rapid advances in genomic and transcriptomic sequencing technologies have enabled the compilation of vast libraries of protein sequences and thus an explosion in bioinformatics-based fields of research. Among these is proteomics – the study of global protein abundance within a biological system. Proteomics has risen to considerable prominence within the biological and health sciences due to its ability to grasp the subtle complexities of protein biochemistry on an impressive, system-wide scale. In conjunction with the compilation of protein-sequence libraries, advances in liquid chromatography and mass spectrometry have allowed for the reliable and rapid deconvolution, identification – and more recently, quantitation – of proteins within complex mixtures.
Majorly comparative in design, modern proteomics experiments aim to aid in our understanding of how biological systems respond to specific conditions. Because of this, most proteomic quantitation is relative, typically being achieved using stable isotopic labels; proteins originating from two separate experimental conditions are independently labelled with heavy or light stable isotope tags, then mixed together in equivalent proportions. Subsequent analysis of the proteins present in the sample by mass spectrometry allows for the direct comparison of proteins’ abundances relative to each other, and inference of causality with respect to the experimental variable. While incredibly elegant in design, such techniques are often impractical, being intensive with respect to cost, time, and sample-handling.
With applications ranging from the study of individual proteins and their biological functions to the complexities of disease pathogenesis, there has recently been a push toward the development of robust methods for label-free comparative proteomic quantitation. As a result, several techniques for quantitative label-free proteomics have been developed, typically relying on one of two strategies for determining a protein’s abundance within a sample; namely spectral counting (counting the number of peptide fragments observed which match a protein’s theoretical fragmentation pattern) and extraction of ions’ absolute intensities (integration of the total abundance of ions determined to correlate with a protein).
However, several caveats exist for each method and its implementation. Proper methods for data correction and normalization, the treatment of missing values between datasets, and statistical testing/correcting procedures all remain contentious and active areas of research. As such, there exists a lack of consensus on which strategies – and their execution – are best.
Yet, due to the practicality and suitability of label-free proteomic quantitation in the study and characterization of nearly any biological system – including those frequented by diagnostic medicine – I have become a strong proponent of its use. This advocacy has led to our development of a robust, reliable, reproducible, and practical approach to label-free proteomic analyses. Through sample-specific normalization in addition to building upon previously proposed techniques, we provide framework for future label-free proteomic studies with application in any of a myriad of biological systems.
This thesis herein explores the development and application of our mass spectrometry-based label-free semi-quantitative comparative proteomics technique, utilizing the sample-specific normalization of proteins’ absolute ion abundances in the characterization of:The proteomic composition of murine hepatic lipid droplets; how they change in response to dietary stress experienced by periods of fasting or fasting followed by re-feeding; and the implications the dynamics of these organellar proteomes may have in their physiological function.
The proteomic changes observed in vivo for EL4-lymphoma tumours either untreated or treated with an etoposide-cyclophosphamide chemotherapeutic cocktail, and the implications these changes may have towards our understanding of tumour-death.
The proteomic differences of luminal-subtype estrogen-receptor positive breast tumours from patients experiencing either disease-free or disease-recurrent survival; the identification of sub-populations of these tumours based on patients’ recurrence status, as defined by protein abundance; and the identification of several proteins potentially predictive of a patient’s disease-free survival.
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- Graduation date
- Fall 2018
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- 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.