Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 199-226). Also available in print.

Thesis (Ph. D.)--Florida State University, 2006. / Advisor: Kenneth D. Weston, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Sept. 19, 2006). Document formatted into pages; contains xv, 96 pages. Includes bibliographical references.

Thesis (Ph. D.)--Florida State University, 2005. / Advisor: Qing-Xiang A. Sang, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed May 11, 2006). Document formatted into pages; contains xvii, 118 pages. Includes bibliographical references.

Thesis (M.S.)--Rochester Institute of Technology, 2005. / Typescript. Includes bibliographical references (leaves 100-104).

The work presented in this dissertation centers around the development of analytical tools for the study of advanced proteomics. Section 1 of this work reviews the need for high efficiency protein separation techniques. Dynamic isoelectric focusing (DIEF) is new technique similar to capillary isoelectric focusing (CIEF) invented by Dr. Luke Tolley at Southern Illinois University Carbondale. Using DIEF, the electric field inside the separation capillary can be modified using high voltage electrodes, additional to the anode and cathode, to control the depth and shape of the resulting pH gradient. By changing the pH gradient, the location and width of focused protein bands can be controlled. As a new analytical technique, the development of DIEF required the design and fabrication of special holders which allow for electrical connections to be made at lengths along the separation capillary. These holders were also designed to have a removable section of capillary to extract very specific pH range proteins from high-resolution separations. Higher throughput DIEF systems were investigated, as well as multiplexed DIEF systems. Section 2 covers the topic of dynamic isoelectric/anisotropy ligand binding assay (DIABLA). DIABLA is a new method used to identify proteins in a complex sample that bind to a known molecule. DIABLA has the potential to be used in two complimentary ways, discovery mode and scanning mode. Both modes are accomplished by using DIEF, followed by fluorescence anisotropy as a sensitive detection method. This allows the entire length of capillary to be scanned to identify areas of non-zero anisotropy, which indicate binding interactions between the protein and target molecule. The binding protein(s) can then be extracted using the removable section of capillary from the DIEF holder, and can be identified by using a second dimension analysis, such as LC/MS/MS. DIABLA was verified in a series of proof-of-concept experiments in both discovery and scanning modes. These experiments involved fluorescently tagging proteins that were focused in the presence of a ligand tagged with a different fluorophore. The usefulness of DIABLA as a separation technique was demonstrated in four specific analyses of complex protein samples in Chapter 10.

There is currently no clinically useful screening test available to identify nulliparous women at high risk of developing pre-eclampsia. This study aimed to identify novel biomarkers using hypothesis generating proteomic methods applied to plasma samples obtained prior to clinical diagnosis of pre-eclampsia. Plasma samples taken at 15 weeks gestation from women who subsequently developed late pre-eclampsia (> 34 weeks), early pre-eclampsia (< 34 weeks) and two distinct groups of women with uncomplicated pregnancies (each n=12) were pooled. Pooled plasma was immunodepleted, labelled using iTRAQ-8 plex reagent and separated into fractions using high pH reverse phase chromatography. Fractions were analysed by LC-MS/MS and data interrogated using ProteinPilot 3.0. The merits of two immunodepletion systems were compared; the Seppro® IgY 14 -SuperMix LC column system removes up to 100 highly abundant plasma proteins and the Multiple Affinity Removal LC column depletes 14 highly abundant plasma proteins. Removal of more high abundance proteins allowed identification of more, potentially interesting, low abundance proteins, but was less reproducible than removing fewer proteins. Two methods of LC-MS/MS analysis were assessed; the QStar XL qTOF and 5800 MALDI-TOF-TOF. The protein identifications and the quantification data acquired by each method was comparable and complementary and increased the total number of proteins identified. A total of 502 proteins were identified. A stringent two stage analysis was developed to identify candidate proteins which changed in abundance in plasma from women who later developed pre-eclampsia compared to women with uncomplicated pregnancies. Analysis identified a total of 113 proteins which were both reproducibly quantified and changed by more than the expected range of biological variation. Six candidate proteins changed in abundance in the plasma taken from women who subsequently developed early pre-eclampsia were selected for further validation. A high throughput, low cost, method of multiple reaction monitoring which allows relative quantitation without the use of costly isotopically labelled peptides was developed to validate candidate proteins. Candidate proteins were also assessed by western blot and ELISA. Only one candidate protein; platelet basic protein, was validated by all three methods and demonstrated similar increases in the abundance. This investigation suggests that measurement of platelet basic protein at 15 weeks gestation is a novel candidate predictive marker for pre-eclampsia. Validation of platelet basic protein in a large, independent, sample set is required to confirm changes in protein expression and to evaluate potential, alongside other factors, to identify nulliparous women at high risk of developing pre-eclampsia later in pregnancy.

Cellular redox homeostasis mediates a wide range of physiological and developmental processes. Various stresses trigger over-production of reactive oxygen/nitrogen species which leads to oxidative modifications of redox-sensitive proteins. Identification and characterization of redox-sensitive proteins are important steps toward understanding molecular mechanisms of stress responses. In the study, a high-throughput quantitative proteomic approach termed OxiTRAQ was developed for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential labeling of reduced and oxidized thiols, and the biotin-tagged peptides are affinity-purified and labeled with iTRAQ reagents for quantitation. This approach allows identification of the specific redox-regulated cysteine residues in proteins and offers an effective tool for elucidation of redox proteomes. With this approach, we identified 195 cysteine-containing peptides from 179 proteins whose thiols underwent oxidative modifications in Arabidopsis cells following the treatment with hydrogen peroxide. A majority of those redox-sensitive proteins, including several transcription factors, were not identified by previous redox proteomics studies. Besides, this method was also used to identify proteins that underwent oxidative modifications in Arabidopsis cells subjected to 15 minute treatment of salicylate (a key signaling molecule in the plant defense pathway) or flg22 (a peptide from bacterial flagellin that induces pathogen associated molecular patterns-triggered immunity). In total, 127 peptides from 111 distinct proteins were identified as salicylate- and/or flg22-responsive redox-sensitive proteins. Among the identified redox sensitive proteins are many regulatory proteins including those involved in chromatin remodeling, transcription, nucleocytoplasmic shutting, and posttranslational regulation. Furthermore, in vivo 15N metabolic labeling method combined with a cysteine-containing peptide enrichment technique was applied to identify proteins that undergo oxidative modifications in plants in response to pathogen attack. The identification of redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding the biological significance of redox signaling in plant stress response.

Micro-organisms are the most abundant and diverse form of life on Earth. Their ability to tolerate stress has enabled them to colonise many inhospitable environments. Microbial processes alter the chemistry of the environment which has left a lasting mark on the geological record. On the other hand, microbial life is heavily influenced by environmental conditions. Indeed, the history of the Earth is shaped by the co-evolution of microbial and geological processes. This thesis explores how micro-organisms are influenced by their environment, with particular reference to microbial rock habitats. Rock habitats are an interesting system to understand the inter-relationship between microbial life and it's environment as they are relatively simple and very common. Rock-dwelling communities are also exposed to numerous stresses such as surface UV exposure, desiccation, temperature fluctuations, low nutrient availability or toxicity from elements leached from the rocks themselves. Three specific aspects of microbial stress in rock environments are investigated here: 1) The use of rocks as a shield from surface UV radiation stress, 2) The microbial response to chemical changes during water-rock interactions, 3) The effect of simultaneous limitation of more than one nutrient. The first uses exposure facilities aboard the International Space Station to provide empirical evidence that colonisation of the early land masses by phototrophs was not inhibited by high surface UV radiation. The latter studies use quantitative proteomics to investigate the cellular response of a heterotrophic bacterium to nutrient deficiency and element leaching, two common stresses in rock habitats. Together these results further our understanding of the relationship between micro-organisms and rocks, both today and over geological time.

All plants are exposed to abiotic and biotic stresses and have developed intricate signalling responses to survive. They respond to cell-structure disruption caused by herbivore probing and feeding by the formation of callose. Callose is a linear homopolymer made up of β-1,3-linked glucose residues with some β-1,6-branches. Plant responses to abiotic or biotic stress share events such as phosphorylation, membrane depolarization, calcium influx and the release of reactive oxygen species such as hydrogen peroxide. These events lead to the up-regulation of several pathways leading to biosynthesis of signalling molecules such as salicylic acid, jasmonate, abscisic acid and ethylene pathways. The aim of this study was to determine the most suitable proteomic approach for identifying proteins and signalling pathways involved in cereal response to aphid infestation. An in silico approach was first evaluated in which the 5ʹ upstream regulatory region of proteins belonging to the family of callose synthases was scanned for cis-regulatory elements in order to identify which callose synthases are possibly expressed in plants during biotic or abiotic stresses. Bioinformatics tools were used in the identification of twelve Arabidopsis and ten rice callose synthase coding regions. Genome sequences for rice and Arabidopsis were scanned for the 2000 bp 5ʹ region upstream of the start codon of each callose synthase coding region. PlantCare, PLACE and Athena software were used to identify putative cis-regulatory elements present in the 2000 bp 5ʹ upstream sequences. The majority of cis-acting elements identified were involved in drought and high temperature responses and only one cis-acting element was involved in wound stress. These results therefore indicated a probable role for plant callose synthases in drought stress responses rather than in biotic stress responses. Genevestigator analysis of Arabidopsis results of micro-array experiments indicated that AtGSL10 is highly up-regulated, with AtGSL1, 3, 5, 6, 7, 8, 11 and 12 showing medium up-regulation and AtGSL2, 4 and 9 no up-regulation during aphid infestation of Arabidopsis plants, implicating a possible role for AtGSL10 in the plant response to aphid infestation. An LC/MS/MS approach was used to identify specific signalling pathways involved in wheat resistance or stress response to aphid infestation. Eight proteins were identified as being up-regulated during aphid feeding in wheat, and 11 proteins were identified as possibly involved in the wheat resistance mechanism against aphid infestation. Several proteins were also identified as constitutively expressed proteins, during normal conditions and aphid infestation. Most pathways identified with proteins up-regulated in the resistance mechanisms of TugelaDN plants, were related to energy metabolism and located in the chloroplast. Evaluation of two dimensional gel electrophoresis to identify phosphoproteins differentially regulated in wheat during aphid infestation, revealed the up-regulation of three proteins namely photosystem II oxygen-evolving complex protein 2, HVUNKNOWN from Hordeum vulgare subsp vulgare and HSKERAT9 NID from Homo sapiens. Additional 57 proteins were partially identified as involved in the stress response but due to low protein levels, the percentage of matching peptides to these proteins was below the required confidence level. Although these protein identifications were below the confidence level, it is interesting to note that several of the proteins are known stress response proteins, and therefore could serve as potential targets for future investigations. In conclusion, the down and up-regulation of wheat proteins after aphid feeding reported in this study suggest that several signalling pathways are involved in the cereal stress response to aphid feeding. In silico approaches require knowledge or identification of potential proteins whereas 2D and LC/MS can identify numerous proteins still unknown to be involved in specific stress responses. The 2D approach is also limited in that the proteins of interest may be in low abundance and therefore not detected in the gels due to the presence of high abundant proteins. Therefore the best approach to identify proteins and signalling pathways involved in the stress response of wheat to aphid infestation, is the LC/MS/MS approach, as this proved to be the most sensitive and robust, identifying the most proteins with a high degree of confidence.

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