Development and application of mass spectrometry for bioanalysis of proteins and metabolites

• Author / Creator

  De Souza, Andrea

• The proteome, a hybrid term from protein and genome, is the total set of proteins expressed by a genome in a cell, tissue, or organism at a given time. Proteomics, the study of the proteome, is concerned with a variety of topics such as identity, quantity, activity, structure, protein-protein interactions, and post-translational modifications. Mass spectrometry is an important tool used in bioanalysis, especially in proteomics research. Recently, proteomics technologies have been applied to toxicology, giving rise to the field of toxicoproteomics. Fish are a valuable model for investigating the impact of toxins on aquatic environments and the zebrafish (Danio rerio) is an excellent candidate for proteomic studies. Gills are an attractive organ for toxicoproteomics, comprising >50% of the total surface area of fish and providing a direct route for the uptake of contaminants. In this work, both qualitative and quantitative mass spectrometry techniques were applied in toxicoproteomics studies. We established the first large-scale proteome profile of a teleost fish tissue using two-dimensional liquid chromatography electrospray ionization tandem mass spectrometry in conjunction with a sequential protein solubilization method for protein fractionation and a precursor ion exclusion method for improving peptide and protein identification efficiency. The identified proteome exhibited excellent coverage of important biochemical pathways relevant to the function of the gill, as well as identifying numerous established and potential biomarkers of stress, disease, and environmental contamination. With an established proteome profile, we utilized a stable isotope labelling strategy to quantify protein expression level changes in the gill upon exposure to two toxicants, naphthenic acids (NAs) and oil sands process water (OSPW), both of which are of environmental concern. Analysis of protein abundance changes in NAs-exposed zebrafish showed reproductive and immune response effects, and OSPW exposures exhibited expression level changes in a vast number of proteins crucial to the structural integrity and function of the gill, as well as proteins involved in immunoregulation and oxidative stress response. In adhering to the use of mass spectrometry in bioanalysis, a method was developed by which urine samples were normalized in an attempt to account for dilution variation, thereby standardizing metabolite concentration measurements among samples.

• Subjects / Keywords

  • Proteomics
  • Metabolomics
  • Mass-spectrometry

• Graduation date

  Spring 2011

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3T34V

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.