<p>Peroxisome is a ubiquitous eukaryotic organelle with a single-layer membrane. It maintains various functions that differ depending on the species and cell types, as well as the environmental or developmental conditions.</p><p>In the first part of this thesis, the peroxisomal protein content was systematically analyzed in different organs in mouse from different ages using proteomic approaches. Thirty-one peroxisomal proteins were identified and ten putative peroxisomal proteins were suggested. The results indicate that peroxisomal proteins show a tissue-specific functional response to the aging process that is probably dependent on their differential regeneration capacity. Besides, alteration in the fatty acid metabolism could alter membrane protein functions; decrease in catalase expression in kidney may contribute to oxidative stress and isoprenoid biosynthesis could contribute to decline in bile salt synthesis. The ability to detect changes in the peroxisomal proteome associated with organ impairment during the course of aging would provide a conceptual framework to understand the role of peroxisome in aging.</p><p>In the second part, peroxisome proteomics was used as a novel approach in marine pollution assessment. The peroxisomal protein expression profiles were obtained and identified from mussel Mytilus sp. exposed to different pollutants, in both laboratory and field experiments. The identified proteins were involved in α- and β–oxidation pathways, xenobiotics and amino acid metabolism, cell signalling, oxyradical metabolism, peroxisomal assembly, respiration and cytoskeleton pathway, etc. Generally, these findings suggest that protein expression signatures could become a valuable tool to monitor the presence of pollutants in marine environment.</p>

<p>Type 2 diabetes mellitus (T2DM) is a heterogeneous disease affecting millions of people worldwide. Both genetic and environmental factors contribute to the pathogenesis. The disease is characterized by alterations in many genes and their products. Historically, genomic alterations have mainly been studied at the transcriptional level in diabetes research. However, transcriptional changes do not always lead to altered translation, which makes it important to measure changes at the protein level. Proteomic techniques offer the possibility of measuring multiple protein alterations simultaneously.</p><p>In this thesis, the proteomic technique surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) has been applied and evaluated in the context of T2DM research. Protocols for pancreatic islet and serum/plasma protein profiling and identification have been developed. In addition, the technique was used to analyze the influence of genetic background versus diabetic environment by determining serum protein profiles of individuals with normal glucose tolerance (NGT) and T2DM with or without family history of diabetes. In total thirteen serum proteins displayed different levels in serum from persons with NGT versus patients with T2DM. Among these proteins, apolipoprotein CIII, albumin and one yet unidentified protein could be classified as being changed because of different genetic backgrounds. On the other hand, ten proteins for instance transthyretin, differed as a result of the diabetic environment.</p><p>When plasma protein patterns of NGT and T2DM individuals characterized by differences in early insulin responses (EIR) were compared, nine proteins were found to be varying between the two groups. Of these proteins five were identified, namely two forms of transthyretin, hemoglobin α-chain, hemoglobin β-chain and apolipoprotein H. However no individual protein alone could explain the differences in EIR. In conclusion, SELDI-TOF MS has been successfully used in the context of T2DM research to identify proteins associated with family history of diabetes and β-bell function. </p>

Mycobacterium avium subsp. hominissuis (MAH) is a pervasive environmental bacterium that can cause opportunistic infections in humans. Among the most robust and hardy members of the Mycobacterium genus, M. avium can persist and thrive in a range of challenging environments, including many which place it in direct contact with humans. Surface-exposed proteins are central to the bacterial processes involved in both environmental persistence and pathogenesis. These proteins also play a critical role in how the immune system of the host recognizes and responds to pathogens. Mycobacteria have evolved a specialized mechanism for protein export, a Type VII Secretion System (T7SS), in order to transport their proteins through their thick and impermeable cell envelope. This system is responsible for the export of several classes of proteins, many of which play an integral role in virulence. A central focus of this dissertation is the characterization of a conserved element of the T7SSs in pathogenic mycobacteria, a PPE family protein, whose deletion attenuates virulence in M. avium. Specifically, we examined the localization of this PPE protein (MAV_2928) within the bacterium, screened potential protein-protein interactions with other conserved elements in the adjacent T7SS loci and analyzed the transcriptional regulation of the gene in response to environmental changes. Seeking to more thoroughly characterize the surface-exposed proteome of M. avium, particularly in the context of early infection, we then developed a method, based on selective biotinylation and affinity purification, to profile the of surface-exposed proteome of the bacterium. We employed this method to analyze the surface-exposed proteomes of M. avium 109 that had been exposed to macrophages to those of M. avium 109 that had been cultured in media. This comparison detected several proteins whose presence at the bacterial surface appeared to be dependent on particular growth conditions. Lastly, in order to establish a more efficient method to isolate biotinylated surface proteins from complex mixtures, we developed a testing paradigm to identify modifications to the original method that might improve our coverage of identified proteins. Through this process, we developed a more robust methodology that yielded improved coverage and depth. We then utilized this technology to profile the surface-exposed proteome of another clinical isolate of M. avium subsp. hominissuis, M. avium 104. Beyond improving our understanding of the basic biology of M. avium, this new data provides independent evidence that PPE family proteins are indeed exported to the surface of M. avium, where they remain associated with the bacterial cell envelope. In total, this analysis represents the most comprehensive profile of the surface-exposed proteins of M. avium generated to date. / Graduation date: 2012

Interest in microfluidic platforms has surged as an alternative for sample preparation in the past two decades, with the potential for miniaturization, portability, automation, integration and parallelism driving this research. However, it is still very challenging to develop an integrated microfluidic device for proteomic preparation for mass spectrometry analysis. My thesis work is focused on the development of a valve-based microfluidic platform interfaced with electrospray ionization mass spectrometry for multiplexed proteomic analysis. First, techniques are developed for the fabrication and packing of multiple beds in a polydimethylsiloxane (PDMS) microdevice, which is compatible with the integration of multilayer microvalves. A soft lithography technique was used to fabricate stable weirs in microchips and new bead introduction techniques were explored for the elimination of bead introduction channels in the design. Such a combination provides a convenient, efficient and effective way for multiple bed preparation in a complex design. Next, detailed studies were carried out on the design parameters and performance of multilayer PDMS microvalves in the presence of high electric fields. These studies guided the integration of electrophoresis methods with valve-based fractionation. Finally, a coupled CE-fractionation-SPE-ESIMS peptide analysis on a totally integrated valve-based microchip is presented. We show the design and operation of a system that performs electrokinetic separation, followed by fractionation into multiple channels, SPE extraction and sample cleanup on packed reaction beds, using a multiplexed, hydraulically valved system, with subsequent mass spectral (MS) analysis. This coupled multiple channel CE-Fractionation-SPE-ESIMS platform on valve-based microchip was successfully applied to peptide analysis.

Mass spectrometry based proteomics has emerged as a powerful methodology for investigating protein expression. “Bottom up” techniques in which proteins are first digested, and resulting peptides separated via multi-dimensional chromatography then analyzed via mass spectrometry provide a wide depth of coverage of expressed proteomes. This technique has been successfully and extensively used to survey protein expression (expression proteomics) and also to investigate proteins and their associated interacting partners in order to ascertain function of unknown proteins (functional proteomics). Azospirillum brasilense is a free-living diazotrophic soil bacteria, with world-wide significance as a plant-growth promoting bacteria. Living within the rhizosphere of cereal grasses, its diverse metabolism is important for its survival in the competitive rhizospheric environment. The recently sequenced genome of strain Sp245 provided a basis for the proteome studies accomplished in this work. After initial mass spectrometer parameter optimization studies, the expressed proteomes of two strains of Azospirillum brasilense, Sp7 and Sp245, grown under both nitrogen fixing and optimal growth (non nitrogen fixing) conditions were analyzed using a bottom up proteomics methodology. Further proteome studies were conducted with A. brasilense strain Sp7 in order to ascertain the effect of one chemotaxis operon, termed Che1. In this study, proteomic surveys were conducted on two bacterial derivative strains, created earlier, which lacked either a forward signaling pathway or an adaptation pathway. The proteomic surveys conducted in this work provide a foundation for further biochemical investigations. In order to facilitate further investigation and a movement into functional proteomics, a set of destination vectors was created that contain a variety of tandem affinity tags. The addition of tandem affinity tags to a protein allow for generic purification schemes, and can facilitate future studies to investigate proteins of interest discovered in the first expression proteomic surveys of A. brasilense. Taken together, this dissertation provides a valuable data set for investigation into the physiology of A. brasilense and further provides biochemical tools for analysis of the functional protein interactions of A. brasilense cells.

Histone modifying enzymes and chromatin remodeling complexes play an important regulatory role in chromatin dynamics that dictate the interaction of regulatory factors involved in processes such as DNA replication, recombination, repair and transcription, with DNA template. The CHD (Chromodomain Helicase DNA Binding Protein) family of proteins is known to be involved in the regulation of gene expression, recombination and chromatin remodeling via their chromatin specific interactions and activities. Phenotypic analysis of the Chd2 mutant mouse model developed by our laboratory indicates that the Chd2 protein plays a critical role in tumor suppression as the heterozygous mutant mice develop spontaneous lymphomas. In this study we demonstrate that mutation of Chd2 renders cells susceptible to inefficient DNA repair and genomic instability. Homozygous and heterozygous Chd2 mutant mouse embryonic fibroblast accumulates higher levels of gamma-H2AX after DNA damage. Chd2 mutant cells show inefficiency in DNA repair of DNA lesions induced by X-rays and UV irradiation as assessed by single cell gel electrophoresis assays. These cells also exhibit increased chromosomal aberrations after treatment with low doses of X-ray irradiation (2 Gy) and show increased radiosensitivity in a clonogenic survival assay. At the molecular level, endogenous CHD2 protein level is induced after exposure to X-ray radiation. In addition, we have also demonstrated in this study that CHD2 is phosphorylated after DNA damage and is a potential substrate for phosphoinositide 3-kinase-related kinases (PIKK) - ATM/ATR. Additionally, mass spectrometric analysis showed possible association of CHD2 with the paraspeckle family of proteins known to be involved in an array of cellular processes specifically in RNA processing and DNA repair. An in vivo splicing assay demonstrated that CHD2 played a role in modulation of pre-mRNA splicing event. Collectively, our findings suggest that CHD2 is a multi-functional protein working with the paraspeckle protein complex to facilitate both the pre-mRNA splicing process and the initial DNA repair process. CHD2 may also be involved in the later stages of DNA damage response pathway by influencing p53’s transcriptional activity.

Signaling lipids such as diacylglycerol (DAG) and the phosphatidylinositol polyphosphates (PIPns) play crucial roles in numerous cellular pathways. However, characterization of their activities is hindered by the complexity of associated signaling pathways and of the membrane environment. To address this issue, we have developed lipid probes that are effective for characterizing biological events using different applications, including activity-based probing (PIPns and DAG) and microarray analysis (PIPns). The activity-based probes have been applied to label receptor targets in multiple cancer cell proteomes through photocrosslinking followed by click reactions. The probes were found to label several proteins, as judged by on-gel fluorescence, and labeling was abrogated through various controls, such as heat denaturation and competition. Proteomic studies have been successfully performed to identify protein targets through biotin enrichment followed by mass spectrometric analysis. For microarray analysis, functionalized PIPn probes were synthesized and applied to develop a high throughput microarray analysis to measure protein-lipid binding affinity. These approaches will be invaluable for characterizing PIPn/DAG-regulated events and their involvement in disease. The design, synthesis and application of these lipid probes are included in this dissertation. In addition, the design and synthesis of other lipid probes are discussed, such as bis(monoacylglycero)phosphate (BMP), and lysophophatidylcholine (LPC) analogs.

Though mass spectrometry has earned a central role in the field of proteomics due to its versatility in a wide range of experiments, challenges and complications are still encountered when using mass spectrometry to characterize protein structures, post-translational modifications (PTMs), and abundances. In this dissertation, analytical methods utilizing mass spectrometry have been developed to address challenges associated with both qualitative and quantitative protein characterization. The effectiveness of using multiple pepsin-like proteases, both separately and in mixtures, combined with online proteolysis using a special triaxial probe has been demonstrated on an amyloid beta peptide related to the onset of Alzheimer’s disease. These findings have broad implications in protein structural characterization studies using hydrogen-deuterium exchange mass spectrometry. A wider range of proteases (Lys-C, Glu-C, and trypsin) and multiple fragmentation methods (collisionally activated dissociation, electron transfer dissociation, and decision tree) have been utilized in the discovery-based PTM characterization of extracellular cellulosome proteins of the bioenergy-relevent organism Clostridium thermocellum, resulting in the identification of 85 previously unknown modification sites in 28 cellulosome proteins. These modifications may contribute to the structure and/or function of the cellulosome protein complex. By using peptide internal standards and a triple quadrupole mass spectrometer operating in selected reaction monitoring mode, a method has been developed for the absolute quantitation of the Clostridium thermocellum cellulosome protein machine in samples ranging in complexity from purified cellulosome samples to whole cell lysates as an alternative to a previously-developed enzyme-linked immunosorbent assay method of cellulosome quantitation. The precision of the cellulosome mass concentration in technical replicates is better than 5% relative standard deviation for all samples, indicating high precision of cellulosome mass concentration for this method. Though methods and results presented in this dissertation have implications in the study of Alzheimer’s disease and bioenergy research, more broadly this dissertation focuses on development of methods to contend with some of the more complex challenges associated with protein characterization currently presented to the field of proteomics.

Proteomics comprises the identification and characterization of the complete suite of expressed proteins in a given cell, organism or community. The coupling of high performance liquid chromatography (LC) with high throughput mass spectrometry (MS) has provided the foundation for current proteomic progression. The transition from proteomic analysis of a single cultivated microbe to that of natural microbial assemblages has required significant advancement in technology and has provided greater biological understanding of microbial community diversity and function. To enhance the capabilities of a mass spectrometric based proteomic analysis, an integrated approach combining bioinformatics with analytical preparations and experimental data collection was developed and applied. This has resulted in a deep characterization of the extracellular fraction of a community of microbes thriving in an acid mine drainage system. Among the notable features of this relatively low complexity community, they exist in a solution that is highly acidic (pH < 1) and hot (temperature > 40°C), with molar concentrations of metals. The extracellular fraction is of particular interest due to the potential to identify and characterize novel proteins that are critical for survival and interactions with the harsh environment. The following analyses have resulted in the specific identification and characterization of novel extracellular proteins. In order to more accurately identify which proteins are present in the extracellular space, a combined computational prediction and experimental identification of the extracellular fraction was performed. Among the hundreds of proteins identified, a highly abundant novel cytochrome was targeted and ultimately characterized through high performance MS. In order to achieve deep proteomic coverage of the extracellular fraction, a metal affinity based protein enrichment utilizing seven different metals was developed and employed resulting in novel protein identifications. A combined top down and bottom up analysis resulted in the characterization of the intact molecular forms of extracellular proteins, including the identification of post-translational modifications. Finally, in order to determine the effectiveness of current MS methodologies, a software package was designed to characterize the > 100,000 mass spectra collected during an MS experiment, revealing that specific optimizations in the LC, MS and protein sequence database have a significant impact on proteomic depth.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 106-113).