Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Wheat is the most agronomically important crop with an annual production of approximately 680 million tonnes per year over the five year period of 2008-2012 (Shewry and Tatham 2016). Wheat typically contributes about 20% of the total calorie intake in Western Europe and between 50-70% in some countries in North Africa and in West and Central Asia. It is estimated that in order to meet the continuous growing global demand wheat production needs to increase by 50% by 2050. Wheat is most commonly consumed as bread, pasta and noodles however it is also used as a food ingredient in other types of foods such as sauces and condiments. The versatility of wheat is largely determined by the unique physiochemical properties of gluten (Bailey 1941). Gluten is one of the earliest proteins to be studied, and was first described by Beccari in 1728 (Bailey 1941) and is readily isolated from wheat flour as a viscoelastic mass. Gluten is a complex mixture of proteins which are the major seed storage proteins found in the cereal grains wheat, barley, rye and oats. Gluten accounts for 70-80% of the total protein content in wheat grains and is traditionally divided into two groups based on their solubility called gliadins and glutenins (Osborne 1907). In genetically pre-disposed patients gluten is able to elicit a non-IgE mediated T-cell response known as coeliac disease (CD). CD affects approximately 1% of the global population for which there is no cure. As no cure is available patients must adhere to a strict gluten-free diet which is often costly and socially excluding. The Codex Standard states that gluten-free foods must contain less than 20 ppm of gluten from wheat, barley, rye and oats and their crossbreeds (FAO/WHO 1983). The Codex Standard also recommends using immunobased methods (or alternative methods) that are able to achieve appropriate sensitivity and specificity for the detection and quantification of gluten with a 10 ppm limit of detection (FAO/WHO 1983). Consequently the current gold standard method for detection of gluten is enzyme linked immunosorbent assay (ELISA) utilising the R5 antibody, however this method is not without shortcomings. Proteomics by mass spectrometry has the potential to offer an alternative, complementary method to determine gluten proteins in foods but for the methodology to become fully validated and accepted it must also overcome similar challenges to immunoassay methods, such as effective extraction of samples and the identification of peptide targets with the requisite specificity. In this research a global approach is taken to aid the development of gluten detection methods using mass spectrometry. One of the major hurdles that has stunted the development of mass spectrometry methods for the detection and quantification for gluten is the lack of protein sequence databases which are required to undertake the MS data searching. In the first results chapter of the thesis a curated gluten protein sequence database was developed (GluPro), and investigated for its utility as a MS data searching tool. It was observed that utilising the GluPro database resulted in improved protein identifications. Following the development of the curated database, extensive method development was carried out to undertake the most extensive background characterisation of the gluten proteome to date using discovery proteomics. To ensure the most comprehensive profile was obtained a number of extraction protocols were investigated and two mass spectrometry platforms with intrinsic differences utilising different modes of acquisition were used. This resulted in the most comprehensive profile of the gluten proteome to date being obtained. In order to meet the continually growing global demand for wheat previous mentioned it is considered that this may be done through the use of genetically modified crops with improved traits such as pesticide resistance. Resulting in the very real possibility of GM crops being introduced into the food supply chain, however there is much widespread public concern regarding the toxicity and allergenicity of genetically modified crops. As wheat is already listed as one of the major eight allergens, it is crucial to be able to undertake safety assessments which are able to assess the toxicity and allergenicity and determine if the GM crop is substantially equivalent to the non-GM counterpart. In the third part of this thesis it is shown how the MS method developed in the previous chapters could be applied and has great potential to be used for safety assessment. Further to this, it is demonstrated how utilising the additional information gathered during the curation of the GluPro database was able to ground the results into in silico measure of toxicity. In the final part of this research all information gathered was interrogated to pick appropriate MRM target peptides, which were unique to a single gluten protein and reproducibly observed to be free from modification. The peptides were synthesised with heavy labels to develop a targeted method to replace current ELISA methods for the detection and quantification. Unexpectedly mass shifts were observed for the precursor ion corresponding to deamidation of the synthetic peptides. Further investigation was undertaken to understand the location and cause of the deamidation sites. This development leads into further recommendation for future development of MRM methods for the detection and quantification of gluten.

Oxysterols are oxygenated derivatives of cholesterol or its precursors. One oxysterol, 24(S),25-epoxycholesterol (24(S),25-EC), which results from a shunt in the cholesterol synthesis pathway has been found at higher than expected levels in embryonic murine brain. Interestingly, the receptor that 24(5),25-EC is a ligand for, Liver X Receptor (LXR), has been implicated in neurogenesis in the ventral mid brain region of embryonic brain; an area with a high density of dopaminergic neurons. The mechanism by which LXR induces this effect is unclear. Therefore, proteomic and phosphoproteomic studies were performed using a stable isotope labelled in amino acid in cell culture (SILAC) approach in order to quantify changes in the proteome between different treatment groups in a mouse substantia nigra dopaminergic cell line (SN4741) SN4741 cells were cultured in SILAC media containing differentially isotope labelled arginine and lysine. For protein expression studies SN4741 cells were treated in serum free media with vehicle, 10muM 24(S),25-EC, or 1muM GW3965, a synthetic ligand of LXR, for 24 hours. For analysis of changes in the phosphoproteome SN4741 cells were treated in serum free media with vehicle, 10muM 24(5),25-EC, or 30muM 25- hydroxycholesterol for 6 hours. Cells were lysed and protein combined in a 1:1 ratio before trypsin digestion and peptide separation via strong cation exchange chromatography. Phosphopeptides were enriched using immobilised metal affinity chromatography (IMAC). Resulting fractions were analysed, using a data dependent LC-MS/MS method. Data was quantified using MaxQuant software in conjunction with Mascot using an IPl mouse database. In protein expression analysis known oxysterol regulated genes, via SREBP or LXR, were differentially expressed. Oxysterol treatment induced global changes in proteins involved in lipid (cholesterol, fatty acid, phospholipid, triglyceride) synthesis. LXR? protein expression increased after GW3965 and 24(5),25-EC treatment, though no change was seen on LXRp mRNA, implying that ligand binding protects LXR? from degradation. 24(S),25-EC induced changes in expression and localisation of the membrane protein caveolin-1. Also, phosphoethanolamine cytidylyltransferase and collagen type IV alpha-3-binding protein, 2 proteins involved in phospholipid synthesis, had an altered expression after 24(S),25-EC treatment suggesting a role for oxysterols in membrane homeostasis. A cytokine, macrophage colony stimulating factor, which is required for normal neuronal development and macrophage differentiation had an LXR independent increased expression after 24(S),25-EC treatment. Quantitative RT-PCR data demonstrated that proteomic changes were due to both transcriptional and post-transcriptional effects of oxysterol. In addition, studies examining changes in the mouse phosphoproteome identified a number of novel phosphorylation sites.

The main work presented in this thesis describes proteomics strategies applied to study the trafficking and turnover of glutathione reductase (Glr1) isoforms in the cytosol and mitochondria of Saccharomyces cerevisiae. Additional work was performed in order to understand mass spectrometric response factors and how they can affect peptides representation in the mass spectra. The opportunity to study two sub proteomes involved in biofilm formation of Pseudomonas aeruginosa PAO1 arose during my PhD and their analysis is also presented. Glr1 is a low abundant protein involved in the defence mechanisms against reactive oxygen species, which are sources of many diseases. Because of its biological relevance, considerable effort has been made in order to understand its functional role in the cell. This protein has been studied using biochemical strategies. In yeast, the cytosolic and mitochondrial forms of glutathione reductase are expressed by the same gene, GLR1, using alternative start codons. Translation from the first AUG codon generates the mitochondrial form incorporating a transit peptide necessary for import into the mitochondria. If the translation starts at the second AUG codon, the cytosolic counterpart is generated. Biochemical approaches show that the first AUG codon is not in favourable context and it has been suggested that leaky scanning accounts for the abundance of the cytosolic protein. The analysis of Glr1 forms by mass spectrometry was demanded because only the N-terminal region is informative about similarities and differences between cytosolic and mitochondrial forms. The protein is also of low abundance in both cytosol and mitochondrial compartments. A genetically modified strain, over-expressing this protein was, therefore, used throughout this work in order to analyse glutathione reductase in the mitochondria. This was not possible with the wild-type strain. Because the first AUG codon is now in context, the over-producing strain (MORF) yields both cytosolic and full length mitochondrial isoforms in the cytosol. Analysis of the mitochondrial protein shows that the cleavage of the pre-sequence is not specific. Three different forms of the mitochondrial N-terminal peptide were detected. Some attention was also devoted to glutathione reductase turnover in both cytosol and mitochondrial compartments using the genetically modified strain. Both N-terminal peptides generated from translation starting in the first and second AUG codon as well as mid-chain peptides from the cytosol fraction and one mid-chain peptide from the mitochondrial fraction, were used to calculate relative turnover measurements. My results illustrate that the mitochondrial protein is in faster turnover than the cytosolic counterpart. Moreover, the long and short forms observed in the cytosol also show slightly different turnover rates, the long form presenting faster turnover than the short form. Rapid turnover therefore maintains the level of glutathione reductase in the mitochondria. Despite the exquisite sensitivity of mass spectrometry, its restricted dynamic range compared with the dynamic range of the entire proteome is limiting for such studies. Peptides have different responses in the mass spectrometry and factors such as hydrophobicity and gas-phase basicity, can also contribute to low detectability of some peptides. To maximise the mass spectrometric response of peptides especially the ones derived from low abundant proteins, is extremely important. My thesis therefore includes a study of mass spectrometric response of peptides generated by different enzymes. Applying the kinetic method, the importance of the position of basic residues on gas-phase basicity and thus on the mass spectrometric response was demonstrated. In addition, the opportunity to carry out a related study on the proteome analysis of membrane vesicles and matrix within biofilms of Pseudomonas aeruginosa PAO1 has arisen and results of this study were presented in the final results chapter. It is the first time that two-dimensional chromatography was applied to analyse these sub-proteomes. Moreover, previous studies were mostly limited to the planktonic population; here the proteomes of membrane vesicles and extracellular matrix with the biofilm were addressed.

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Glomerular filtration is a vital physiological process removing waste products from the circulation and this process occurs across the glomerular filtration barrier (GFB). The cells and extracellular matrix (ECM), which form this barrier, are exposed to forces during ultrafiltration and special adaptation is required to withstand these forces. Dysfunction in cellular adhesion machinery or ECM assembly within the GFB causes loss of selective glomerular filtration, however, the mechanisms governing these processes are poorly understood. To this end we sought to characterise the glomerular ECM and adhesion machinery using high throughput mass spectrometry (MS)-based proteomics. MS of human glomerular ECM identified a highly complex extracellular niche, revealing the potential involvement of novel ECM proteins in glomerular development and disease processes. Furthermore we identified that glomerular cells in culture had distinct ECM proteomes and interestingly, coculture experiments demonstrated that the ECM proteome was influenced by cellular crosstalk and had a closer resemblance to glomerular ECM in vivo. Protein network analyses of in vivo and in vitro ECM datasets revealed a common core of highly connected structural ECM proteins that may be important for glomerular ECM assembly. To understand how this ECM proteome altered in disease, we studied mice with mild glomerular dysfunction. Here, transcriptomic and proteomic analyses identified alterations in ECM composition and 3D electron microscopy revealed striking ultrastructural changes in glomerular ECM. MS-based proteomics was next applied to the analysis of glomerular podocyte adhesion complexes, leading to the discovery that the actin cytoskeletal regulators and trafficking machinery are recruited to adhesions sites in an ECM-ligand dependent manner. Furthermore, these differences functionally altered cell shape and adhesion strength. These same analyses were applied to podocyte cell-cell junctions, revealing an unexpected overlap of cell-ECM and cell-cell adhesion machinery. Overall, these findings demonstrate for the first time the complexity of the glomerular ECM and adhesion signalling complexes and reinforce the benefits of global, unbiased experimental approaches. In addition the results suggest that glomerular ECM composition, organisation and adhesion signalling are context dependent, and therefore, represent potential therapeutic targets.

Protein phosphorylation governs many regulatory pathways and an increasing number of kinases, proteins that transfer phosphate groups, are in turn activated by cyclic nucleotides. One of the cyclic nucleotides, cyclic adenosine monophosphate (cAMP), has been shown to be a second messenger in abiotic and biotic stress responses. However, little is known about the precise role of cAMP in plants and in the down-stream activation of kinases, and hence cAMP-dependent phosphorylation. To increase our understanding of the role of cAMP, proteomic and phosphoproteomic profiles of Arabidopsis thaliana suspension culture cells were analyzed before and after treatment of cells with two different concentrations of 8-Bromo-cAMP (1 µM and 100 nM) and over a time-course of one hour. A comparative quantitative analysis was undertaken using two- dimensional gel electrophoresis and the Delta 2D software (DECODON) followed by protein spot identification by tandem mass spectrometry combined with Mascot and Scaffold. Differentially expressed proteins and regulated phosphoproteins were categorized according to their biological function using bioinformatics tools. The results revealed that the treatment with 1 µM and 100 nM 8-Bromo-cAMP was sufficient to induce specific concentration- and time-dependent changes at the proteome and phosphoproteome levels. In particular, different phosphorylation patterns were observed overtime preferentially affecting proteins in a number of functional categories, notably phosphatases, proteins that remove phosphate groups. This suggests that cAMP both transiently activates and deactivates proteins through specific phosphorylation events and provides new insight into biological mechanisms and functions at the systems level.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most abundant environmental pharmaceutical contaminants. In this study, a proteomic analysis was conducted to identify proteins differentially expressed in gill tissue of zebrafish (Danio rerio) after a 14-day exposure to the NSAIDs ibuprofen or naproxen. A total of 104 proteins with altered expression as indicated by 2-dimensional electrophoresis were analyzed by liquid chromatography with ion trap mass spectrometry (MS/MS). A total of 14 proteins fulfilled our requirements for identification which included consistency among replicate gels as well as successful MS/MS ion searches with the MASCOT database. The most prominent feature of the differential protein expression observed after NSAID exposure was an up-regulation of proteins belonging to the globin family which are involved in the transport of oxygen from gills and availability of heme molecules required for synthesis of cyclooxygenase. Differential expression was observed at exposure concentrations as low as 1-10 µg/L indicating that altered gene expression may occur in fish subjected to environmentally realistic levels of NSAID exposure.

Thesis advisor: Eranthie Weerapana / Protein activity is modulated by hundreds of post-translational modifications (PTMs). This thesis will describe the development and application of proteomic methods to study three chemically distinct PTMs. In the first project we describe the development of a proteomics platform to identify cysteine oxidation sites on interactors of the NADPH oxidase complex in response to EGF activation. The NADPH oxidases (Nox) are the source of H(2)O(2) which acts as a secondary messenger during EGFR activation. Known targets of Nox H(2)O(2) include phosphatases PTP1B and PTEN. Oxidation of the active site of PTP1B and PTEN temporarily inactivates their phosphatase activity which allows for EGF signal propagation. The platform involves combining TurboID with OxICAT to identify proteins which are oxidized by Nox2 in a spatially and temporally controlled manner. In the second project, our goal is to identify proteins which recognize two Met oxidation sites in actin known to play a role in regulating the transition between F and G actin. We utilized a peptide based photo-crosslinking approach to identify PFKL and HSP70s HSPA8 and HSPA1B as putative "readers" of oxidized or unoxidized methionine in actin respectively. Finally, protein citrullination is a enzyme catalyzed PTM where the guanidinium on arginine is converted into a urea by a family of enzymes called protein arginine deiminases. Aberrant citrullination is linked to many human diseases including rheumatoid arthritis. Therefore, proteomic methods to characterize citrullination can provide insights into disease pathophysiology. We describe the identification of novel protein targets of with a chemoselective biotin phenyl glyoxal probe, and the development of a label free proteomic method to identify sites of citrullination. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.