No description available.

No description available.

No description available.

Many techniques exist for protein analysis including gel electrophoresis, immunoassays and mass spectrometry to name a few. Although each of these techniques offers different advantages there are challenges associated with protein analysis that present limitations and drawbacks for each technique. These include limited sample volumes, incompatible matrices, complex mixtures, limit of detection, and complicated and time consuming analysis techniques. The research described in this thesis deals with methods which address the above mentioned challenges. The fabrication of enzymatic microreactors and the development of an extraction technique to remove proteins from incompatible matrices are presented. A low temperature solvent bonding technique was developed that can easily be modified to accommodate pH and temperature sensitive enzymes such as pepsin. Microfluidic chips fabricated with poly(methyl methacrylate) were constructed using this bonding method. The low temperature bonding method allows enzymes to be patterned on microfluidic devices prior to bonding with no negative impact on enzyme activity. Both peptic and tryptic reactors were fabricated using this technique. These devices have a lifetime of one month and can perform protein digestions in as little as 2.4 seconds. Alternatively, enzymatic microreactors were developed using a novel enzyme immobilization method on porous polymer monolith columns. These columns have the advantage of having the dual functionality of an enzymatic microreactor and an electrospray emitter permitting on-line digestions to be performed. Furthermore, the new immobilization method allows columns to have a longer lifetime as this method permitted the regeneration of the columns with fresh enzyme once enzyme activity was lost. To overcome the issues associated with incompatible matrices, an extraction technique using acetonitrile and octyl-β-D-glycopyranoside was developed to remove proteins from organic matrices. This was developed to detect the presence of prion proteins in biodiesel. Afterwards, the prion proteins were enzymatically digested and detected by mass spectrometry. Finally both types of enzyme microreactors were applied to proteins that are difficult to digest with traditional in-solution digestions. The digestion of prion proteins and α-1-protease inhibitor were found to be more efficient and conducted in significantly less time demonstrating the potential use of these devices in clinical research. / Thesis (Ph.D, Chemistry) -- Queen's University, 2011-10-02 19:11:35.576

No description available.

No description available.

No description available.

No description available.

In this project, “selected ion flow tube mass spectrometry” (SIFT-MS), a sensitive analytical technique, reveals potential for the development of applications in the environment and petroleum areas. Many prior applications have shown their potential for analyzing samples in widely disparate areas. Its fast analysis process and high sensitivity gives it a significant advantage over more conventional methods. This project is directed at expanding this technology to more applications in the petroleum and air quality areas. The application to the petroleum industry has shown that SIFT-MS can quantify H2S and CH3SH in natural gas to 11.8 and 1.2 ppbv, respectively. The SIFT-MS results showed a good linear response with increasing sulfide concentrations by using the H3O+ reagent ion to quantify H2S, CH3SH, and the total combined concentration of DMS and C2H5SH. The ability to use the SIFT-MS instrument to trace chemical tracers, such as bromobenzene and chlorobenzene in hydrocarbon mediums, was also investigated. SIFT-MS showed also the capacity to trace these compounds in natural gas and LPG. The limits of detection (LOD) were also obtained. This study furthermore, found the utility of the NO+ reagent ion to analyse qualitatively some of the large hydrocarbons. Unfortunately, however, the SIFT-MS reactions could not distinguish between the structural isomers of these aromatic and aliphatic hydrocarbons and there was probable conflict between the fragmentation product ions with smaller hydrocarbons. From the air quality perspective, the SIFT-MS also proved its potential for use in air monitoring, using passive techniques and particularly for BTEX (benzene, toluene, ethyl benzene, and xylene) compounds. The study illustrated SIFT-MS’s ability to deal with thermal desorption and passive methodology in general. Ecan (Environmental Canterbury) routinely examines environmental pollutants in Christchurch air by passive sampling methodologies. In this study, we compared and achieved agreement by comparing the result of thermal desorption-SIFT-MS (TD-SIFT-MS) of Christchurch air with the more conventional methodologies of TD-GC-MS and the Ecan agency measurements.

Gap junctions are communicating junctions between cells that allow small molecules to pass from the cytoplasm of one cell to the cytoplasm of an adjacent cell. The pores of gap junctions are comprised of two adjacent connexons on neighboring cells, and each connexon is comprised of six connexin proteins. The eye lens of vertebrates is an avascular tissue that is dependent on gap junctions for the distribution of nutrients as well as the removal of waste products. In addition, as the lens cells develop into fibers, they lose their intracellular organelles including the membrane-bound organelles, and are highly dependent on connexons for movement of metabolites and waste materials. Only two connexins, in Bos Taurus Cx44 and Cx49, are highly expressed in lens fiber cells. Thus, the lens offers an excellent system for studying gap junctions. In this study, high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) techniques were used to isolate and characterize connexin proteins from the eye lens of the cow and mouse. Despite over 300 proteins being identified from bovine lens using MS techniques, it was still possible to identify the two connexin proteins following proteolytic digests and MS analysis of the resultant peptides. Several post- translational modifications (PTMs) were identified and characterized in lens fiber connexins, including phosphorylations, acetylations and deamidations and proteolytic cleavages. Changes in phosphorylation of several other lens proteins upon the activation of protein kinase C were also identified. Detection of the orthologous proteins in mouse lens proved more challenging, but peptides derived from both connexin proteins were also detected from this tissue and PTMs of mouse connexins were also observed. Glutathione-S-transferase fusions to mouse Cx44 and Cx50 were used to identify a number of proteins that may interact with the mouse connexins, and the relevance of those interactions was considered. The utility of mass spectrometry to the identification of specific proteins from complex mixtures was clearly demonstrated, and its application to understanding the functional relevance of PTMs was discussed.