Hepatic Proteome Analysis of Fathead Minnow (Pimephales promelas) Exposed to Municipal Wastewater in the Bow River

Abstract

Large quantities of contaminants enter Canadian waterways through municipal wastewater effluent (MWWE) each year. MWWE exposure can have a myriad of effects on aquatic organisms, including inducing chronic stress response or altering hormone signaling. A key site for many of these effects is the liver, which controls xenobiotic metabolism, energy regulation, and other functions related to the maintenance of homeostasis. One method for analyzing changes in liver function is shotgun proteomics, which uses mass spectrometry data to identify and quantify proteins in a tissue or sample. The objective of this thesis is to investigate changes in the liver proteome in fathead minnows caged upstream and downstream of wastewater treatment plants (WWTPs) in the Bow River, Alberta.

To effectively analyze mass spectrometry data generated for this project, a fathead minnow specific protein database was constructed from the draft fathead minnow genome and corresponding annotations. The constructed database is compared to the zebrafish reference proteome and UniProt Cyprinidae proteins to determine differences between databases and the effect on protein identification rates. Additionally, several different search engines, including \emph{de novo} and database search engines, are compared against different datasets to determine how mass spectrometry equipment and database accuracy affects protein identification rates. Comparison of the databases showed that a species-specific database provided substantial increases in protein identification rates, with 461 (14.2\%) more proteins identified than from the zebrafish reference proteome and Uniprot database. Search tool comparisons revealed that while \emph{de novo} search engines can increase protein identifications within low quality databases, they are outperformed by standard database search engines when an accurate and comprehensive database is available.

Shotgun proteomics was used to analyze changes in liver proteome of fathead minnows caged in five sites along the Bow River around Calgary, Alberta, Canada. Sites were located upstream, downstream, and close to the outflow of wastewater treatment plants (WWTPs) along the river. The constructed database and a TMT-labelled fathead minnow mass spectrometry dataset are used to quantify protein expression changes in the proteome. Differential expression analysis and gene set enrichment analysis are used to compare expression profiles among different sites. 3689 proteins were identified in the fathead minnow proteome. Differential expression shows large changes in the liver proteomes of fish located near the outflow of the Bonnybrook WWTPs, with a similar, but reduced effect at sites further. Proteins and gene sets in involved in lipid metabolism, oxidative stress, xenobiotic removal were upregulated, while mRNA splicing, and cytoskeleton organization were downregulated. Fish near the outflow also showed changes in cell cycle control and protein modifications compared to fish further downstream.

Identifying changes in the liver proteome of MWWE-exposed organisms is important for identifying potential biomarkers and understanding how exposure affect the health of aquatic organisms. Changes in peroxisomal lipid metabolism and mitochondrial proteins suggest mitochondrial activity for further study of the impacts of MWWE. Additionally, analysis of the impacts of species-specific database on shotgun proteomics is useful for the application of omics to non-model organisms. Comparison of the different database shows that even first draft genome sequences can produced substantially improved protein identification rates over protein databases derived from other organisms.