published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

 Proteomics is the systematic study of the proteome: the total protein expression of a cell or tissue under specified conditions. The multiplicity and complexity of proteins in cells requires sensitive, selective, and comprehensive methodologies for their distinction and characterization. Multidimensional liquid chromatography (MDLC) coupled with biological tandem mass spectrometry (MS/MS) is uniquely suited to fulfill those requirements and has become an indispensable tool in MS-based proteomics. Our laboratory has developed an online high-/low-pH reversed-phase/reversed-phase (RP–RP) LC system exhibiting fully automatable and reproducible performance. It is a promising alternative to the strong cation exchange/reversed-phase (SCX–RP) system commonly used in high-throughput comprehensive proteomics analyses. The first part of this Thesis (Chapter 2) describes the development of a variant of the high-/low-pH RP–RP platform—RP–SCX–RP—that integrates an additional SCX trap column between the two RP columns to enhance sample recovery. This new system allows the detection of larger numbers of hydrophilic peptides. Indeed, in the analyses of a lysate of Arabidopsis chloroplast proteins, it identified approximately 25% more non-redundant proteins than those identified using the previous version of the RP–RP system. The modified platform has been extended for the online removal of sodium dodecyl sulfate and other excess interference chemicals used in Isobaric Tags for Relative and Absolute Quantification (iTRAQ) reactions, thereby avoiding the need for time-consuming offline SCX clean-up prior to RP–RP separation in the quantitative proteomics analyses of crude biological samples at low-microgram levels. A novel online three-dimensional liquid chromatography (3DLC) system was derived from the RP–SCX–RP design, exhibiting remarkably enhanced orthogonality, resolution, and peak capacity. Peptides were separated in the first-dimension high-pH RP column based on their hydrophobicity, followed by sub-fractionation in the second-dimension SCX column, primarily based on charge; the third dimension was a typical low-pH RP separation, prior to MS analysis. The overall performance of the system was evaluated through analysis of a cell lysate of mouse embryonic fibroblasts. Relative to the two-dimensional high-/low-pH RP–RP system, the new 3D system yielded significant increases in the number of unique peptides and proteins identified, making it a good alternative to SCX–RP and high-/low-pH RP–RP as an efficient automated MDLC platform for high-throughput shotgun proteomics. An optimized and miniaturized variant of the three-dimensional LC platform was also developed. Its simplified setup and operation, by decreasing the number of six-port switching valves (from three to two) and the number of SCX fractionation steps, minimized both the potential sample loss and the total analysis time (by ca. 30%). Thus, a variety of novel, automatable, and robust RP–SCX–RP-based MDLC platforms have been developed for high-throughput qualitative and quantitative analysis. The performance of these systems complements conventional MDLC systems, with enhanced quality, quantity, reproducibility, and throughput of protein identification and quantification. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Paralytic shellfish poisoning (PSP) and ciguatera fish poisoning (CFP) are the two major contributors to illnesses caused by dinoflagellate toxins. Paralytic shellfish poisoning toxins (PSTs) are produced by dinoflagellates in the genera Alexandrium, Gymnodinium, and Pyrodinium while ciguatera fish poisoning toxins, such as ciguatoxins (CTXs), are originated from benthic toxic dinoflagellates (Gambierdiscus, Prorocentrum, Ostreopsis, and Coolia species). These toxins are responsible for human intoxication syndromes to the nervous system and muscles. This study optimized the protein extraction for proteomics analysis of dinoflagellate. The protein and toxin profiles of Alexandrium tamarense CI01 at different toxin biosynthesis phases were compared; differentially expressed proteins in highly toxic algae were identified using matrix-assisted laser desorption/ionization time-of–flight (MALDI-TOF) spectrometry. Some potential proteins involved in the toxin biosynthesis of A. tamarense CI01 were also identified. The protein preparation method from dinoflagellates for proteomics is selective for proteins with different pIs and molecular weights. The Lysis method may cause variation of the target proteins, but Trizol and Tris do not. Trizol method is good at analyzing hydrophobic, high molecular weights or binding proteins on the membrane. These PST synthesis-related enzymes are common in dinoflagellates, thereby 2-DIGE and Trizol are the best staining methods for dinoflagellates toxin synthase proteomics research. Through a comparative study in A. tamarense CI01 under different nutrient conditions and growth phase, the potential toxin synthesis-related proteins were investigated. Based on proteomics results, methionine, ornithine, arginine metabolism-related proteins and photosynthesis-related proteins may be related to the PSTs biosynthesis. Results also identified a similar pathway of PST biosynthesis in both dinoflagellate and cyanobacteria. A comparative proteomics study was applied to identify proteins of biomarkers for CTX accumulation in hepatic tissue of grouper, Cephalopholis argus, and PSTs accumulation in shellfish, Saxidomus giganteus. In C. argus with elevated CTXs, ATP synthase subunit beta and cytochrome c, ubiquitin enzymes, ATP related enzymes, and telomerase reverse transcriptase were greatly reduced. The comparative proteomic analysis revealed that PSTs and CTXs induced influx/efflux of Na+ or Ca2+ disorders in fish and shellfish. ATP synthase can control the concentration and influx/efflux of Na+. Alterations of Na+/K+ adenosine triphosphatase, liagand gated ion channel and sarcoplasmic calcium-binding protein in toxic shellfish was believed to reduce damage that PSTs (sodium channel blockers) bring to shellfish by controlling the concentration and influx/efflux of Na+ or Ca2+. Two identified resistant mechanisms to Na+ channel toxins are amino acid mutation and toxin affinity-binding proteins. However, neither of them was confirmed in the present study, but the proteins controlling the concentration and influx/efflux of Na+ or Ca2+ in this study may be due to new mechanisms. In conclusion, our comparative proteomic analysis revealed that CTXs and PSTs induced influx/efflux of Na+ or Ca2+ changes in toxic samples with a concomitant interference with signal transduction, metabolomics processes, detoxification, and anti-apoptosis. The physiological roles of ion concentration controlling and ion signal-related proteins in toxic fish and shellfish species should be further studied for their potential roles in resistance mechanisms to CTXs and PSTs. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

There continue to be great strides in the field of proteomics but as samples become more complex, the ability to increase sequence coverage and confidence in the identification becomes more important. Several methods of derivatization have been developed that can be used in combination with tandem mass spectrometry to identify and characterize proteins. Three types of activation, including infrared multiphoton dissociation, ultraviolet photodissociation, and electron transfer dissociation, are enhanced in this dissertation and compared to the conventional method of collisional induced dissociation (CID) to demonstrate the improved characterization of proteins. A free amine reactive phosphate group was synthesized and used to modify the N-terminus of digested peptides. This phosphate group absorbs at the IR wavelength of 10.6 µm as well as the Vacuum-ultraviolet (VUV) due to an aromatic group allowing modified peptides to be dissociated by infrared multi-photon dissociation (IRMPD) or ultraviolet photodissociation (UVPD) whereas peptides without this chromophore are less responsive to IR or UV irradiation. The PD spectra for these modified peptides yield simplified MS/MS spectra due to the neutralization of all N-terminal product ions from the incorporation the negatively charged phosphate moiety. This is especially advantageous for UVPD due to the great number of product ions produced due to the higher energy deposition of the UV photons. The MS/MS spectra also produce higher sequence coverage in comparison to CID of the modified or unmodified peptides due to more informative fragmentation pathways generated upon PD from secondary dissociation and an increased ion trapping mass range. IRMPD is also implemented for the first time on an orbitrap mass spectrometer to achieve high resolution analysis of IR chromophore-derivatized samples as well as top-down analysis of unmodified proteins. High resolution/high mass accuracy analysis is extremely beneficial for characterization of complex samples due to the likelihood of false positives at lower resolutions/accuracies. For electron transfer dissociation, precursor ions in higher charge states undergo more exothermic electron transfer and thus minimize non-dissociative charge reduction. In this dissertation, cysteine side chains are alkylated with a fixed charge to deliberately increase the charge states of peptides and improve electron transfer dissociation. ETD can also be used to study protein structure by derivatizing the intact structure with a hydrazone reagent. A hydrazone bond will be preferentially cleaved during ETD facilitating the recognition of any modified residues through a distinguishing ETD fragmentation spectrum. / text

Cardiovascular disease (CVD) is the leading cause of death in the United States. Oxidative stress plays an important role in the pathogenesis of CVD. Heart failure is the end point of many forms of CVD. The purpose of this study is to identify novel cardiac specific indicators of oxidative injury useful for early and convenient diagnosis of heart failure.To determine the most suitable method for identification of non-invasive oxidative injury indicators in general, human diploid fibroblasts (HDFs) were treated with H2O2 for collection of mRNA, cell lysates and conditioned media to perform cDNA microarray and LC-MS/MS based Multidimensional Protein Identification Technology (MudPIT) analyses. Electron Spray Ionization (ESI)-LC-MS/MS analysis of the conditioned media led to the finding of IGFBP-6 as a non-invasive biomarker of cell oxidative injuy in vitro and in vivo. The data obtained from this study indicate that proteomic analysis of conditioned media is useful to identify non-invasive biomarkers valuable for diagnosis or management of diseases.Cardiomyocyts (CMCs) and Cardiac fibroblasts (CFs) in culture were used to identify cardiac specific indicators of oxidative stress. Increased level of Cystatin C was detected in the conditioned medium of CMCs due to H2O2 treatment. In vivo models of oxidative stress were used to validate the increase of Cystatin C. Cystatin C levels increased in the plasma of mice with doxorubicin induced cardiomyopathy and coronary artery occlusion induced myocardial infarction (MI). These data indicate that Cystatin C can be a potential indicator of CMC oxidative injury in vitro and in vivo.Cystatin C is a cysteine protease inhibitor. The finding that oxidative stress induces Cystatin C led us to investigate a novel pathway regulating cardiac extracellular matrix (ECM) with CFs in culture, increased levels of ECM protein and decreased levels of Cathepsin B (CTB) protein and activity were detected upon Cystatin C treatment. With coronary artery occlusion induced MI mouse model, increased levels of Cystatin C and ECM protein and decreased levels of CTB protein and activity were detected in the infarcted area of the myocardium. These data indicate that Cystatin C serves as a potential fibrotic factor during myocardial remodeling.

During early development of Xenopus laevis, gastrulation is a key morphogenetic event which transforms the embryo into three of primary germ layers: ectoderm, mesoderm, and endoderm. In order for the physical separation of these layers to occur, cells have to acquire specific properties that distinguish one layer from another. These properties, which include cell adhesion and migration, should be reflected in the tissue-specific proteome. While genetic analysis has led to the determination of a number of proteins involved in germ layer formation, this method would not have identified those proteins regulated on a translational or post-translational level. In this study, we have developed a two-dimensional gel based comparative proteomic approach employing difference gel electrophoresis (DiGE) to identify proteins involved in germ layer morphogenesis during Xenopus gastrulation. Differences between the physical properties of the ectoderm and mesoderm are likely based on differences in the proteomes of the cell surface and/or cortex. We therefore analyzed plasma membrane enriched fractions, obtained using discontinuous sucrose density gradient centrifugation. The Decyder program was used to quantify expression changes with statistical confidence across multiple DiGE gels, provide independent confirmation of distinct expression patterns from the individual experiments, and demonstrate high reproducibility between replicate samples. The identity of 23 proteins, which were obtained from 33 analyzed spots, was determined using mass spectrometry. Our proteomic analysis of Xenopus ectoderm and mesoderm identified alterations in proteins involved in cytoskeletal organization, signal transduction, protein folding, vesicle trafficking, and in glycolysis. We have also demonstrated the feasibility of proteomics in Xenopus, and have therefore shown that proteomics may be a valuable tool for analysis of early development in this system.

Hepatitis C virus (HCV) infects an estimated 200,000 people in Canada, and is the leading cause of liver transplants in North America. Viral infection usually leads to chronic infection, and complications include liver fibrosis, steatosis and hepatocellular carcinoma (HCC). The HCV non-structural proteins 3 and 4A (NS3/4A), is a multifunctional protein complex with roles in RNA replication and polyprotein processing. Additionally, the NS3 protease has been shown to induce advanced cellular transformation in vivo and tumour formation in nude mice. However, the mechanism by which transformation occurs remains unknown. The objective of this study was to determine if the naturally occurring NS3/4A protein complex, rather than the NS3 protease domain on its own, could also induce cellular transformation and to determine the changes that NS3/4A expression had on the host cell proteome.

Hypertension is a major risk factor for developing cardiovascular, renal and nervous system pathologies. Refractory hypertension is prevalent at approximately 30% despite diet, lifestyle and multiple pharmacologic interventions. Blood pressure can be influenced at the level of the nervous system, at the level of the kidney, and at the level of the vasculature. This document describes investigations performed into each of these systems during the development of hypertension using a model of human essential hypertension, the spontaneously hypertensive rat (SHR), by a proteomic approach of 2-dimensional gel electrophoresis and mass spectrometry. A transgenic mouse model with deficits in p75 neurotrophin receptor (NTR), which has been implicated in hypertension, was similarly investigated. Proteomics is a scientific strategy that is not entirely hypothesis driven, rather it has the hypothesis that functional and phenotypic changes in pathophysiology are reflected at the protein level. Proteomics compares the proteome of a disease state, treatment, temporal state, to an appropriate control to determine changes in the level of protein present and its biochemical properties. Proteome changes in the SHR mesenteric arteries included the novel observation of increased CLIC1 association with the adventitia in the SHR, the decreased expression of HSP90 alpha and beta in the SHR, and the presence of an additional protein spot for tropomyosin beta and MLC20 in the SHR. These proteome changes are suggestive of an increased contractile state in the mesenteric resistance arteries of SHR. In the renal proteome, there was a decrease of alpha-2μ globulin and a decrease in MAWDBP in the SHR demonstrating molecular changes in the kidney before known pathological changes. In superior cervical ganglia, sstereological measurement showed significant increases in the number of sympathetic neurons in both lines of p75NTR-deficient mice, relative to wild type mice with an enhanced survival of sympathetic neurons associated with shifts toward the more basic isoforms of Annexin V in the proteomes of p75NTR-deficient mice. / Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2014-02-11 15:35:00.525

Proteome profiling is the identification and quantitation of all proteins in biological samples. An important application of proteome profiling that has received much attention is clinical proteomics, a field that promises the discovery of biomarkers that will be useful for early diagnosis and prognosis of diseases. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from complex biological fluids and (ii) extensive biochemical processing (reduction, alkylation and enzymatic digestion) prior to analysis. However, the lack of standardized sample handling and processing in proteomics is a major limitation for the field. The conventional macroscale manual sample handling requires multiple containers and transfers, which often leads to sample loss and contamination. For clinical proteomics to be adopted as a gold standard for clinical measures, the issue of irreproducibility needs to be addressed. A potential solution to this problem is to form integrated systems for sample handling and processing, and in this dissertation, I describe my work towards realizing this goal using digital microfluidics (DMF). DMF is a technique characterized by the manipulation of discrete droplets (100 nL – 10 L) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. This thesis demonstrates how DMF can be a powerful tool capable of automating several protein handling and processing steps used in proteomics.

The role played by the protein UnkG from the plant growth-promoting bacterium Pseudomonas putida UW4 in the ability of the bacterium to facilitate plant growth was studied. Previous work showed that over-expressing UnkG decreased the ability of P. putida UW4 to facilitate plant growth. In contrast, an unkG knock-out mutant of P. putida UW4 displayed an increased ability to promote plant growth. Various biological activities of P. putida UW4, P. putida UW4/pETP and P. putida UW4/pETP-unkG have been compared. Thus, the growth curves were measured; the Biolog™ system was used to test the ability of these strains to utilize various carbon sources; the strains were observed by scanning electron microscopy to assess their relative cell sizes; biochemical assays were conducted to quantify 3-indoleacetic acid production and to measure the enzymatic activity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase; proteome-level changes of P. putida UW4/pETP and P. putida UW4/pETP-unkG were profiled using two-dimensional difference in-gel electrophoresis (DIGE), followed by mass spectrometry identification of the altered proteins. After running DIGE, sixteen altered proteins were identified and their possible roles in the interactions between the bacterium and plants were discussed. Based on the preliminary results, we hypothesize that 1) UnkG may be detrimental to plant growth; 2) UnkG may negatively regulate a number of key cellular functions in a general way related to the energy balance of the bacterium.