Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Murray V. Johnston, III, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

No description available.

Graduation date: 2001

Graduation date: 1998

Thesis (M.S.)--Oregon Graduate Center, 1972.

Reversible protein phosphorylation is an essential regulatory mechanism used by eukaryotes to coordinate the biochemical processes of the cell. The PP2A phosphatase functions to dephosphorylate specific proteins originally targeted by stimulus-activated protein kinases. The identification of a sub-network surrounding the human PP2A-Striatin holoenzyme, termed STRIPAK, provides insight into novel mechanisms for PP2A function and regulation. I reveal that STRIPAK participates in at least two mutually-exclusive sub-complexes, one of which contains the putative cortactin-binding protein, CTTNBP2NL. I show that CTTNBP2NL is enriched at the actin cytoskeleton, likely in a STRIPAK-independent manner. This study also reveals that STRIPAK interacts with a subunit of the dynein motor, at least partially, through CTTNBP2NL. This work will serve as a platform for the structural and functional characterization of STRIPAK and will ultimately assist in defining novel mechanisms of regulation and function for the human PP2A phosphatase.

Reversible protein phosphorylation is an essential regulatory mechanism used by eukaryotes to coordinate the biochemical processes of the cell. The PP2A phosphatase functions to dephosphorylate specific proteins originally targeted by stimulus-activated protein kinases. The identification of a sub-network surrounding the human PP2A-Striatin holoenzyme, termed STRIPAK, provides insight into novel mechanisms for PP2A function and regulation. I reveal that STRIPAK participates in at least two mutually-exclusive sub-complexes, one of which contains the putative cortactin-binding protein, CTTNBP2NL. I show that CTTNBP2NL is enriched at the actin cytoskeleton, likely in a STRIPAK-independent manner. This study also reveals that STRIPAK interacts with a subunit of the dynein motor, at least partially, through CTTNBP2NL. This work will serve as a platform for the structural and functional characterization of STRIPAK and will ultimately assist in defining novel mechanisms of regulation and function for the human PP2A phosphatase.

Analyzing the chemical compounds in biological fluid, such as human blood, by mass spectrometry, sample pretreatments are frequently needed. Those methods, including centrifugation, solid phase extraction (SPE) and liquid-liquid extraction, separate and remove the interferences in biological samples prior to mass analysis. The drawbacks of sample pretreatments mentioned above are time consuming and tedious operation procedures. Thin layer chromatography (TLC) is an analytical tool commonly used for sample purification and separation. To characterize the chemical compounds on TLC plate, coupling TLC with mass spectrometry seems to be a most efficient approach. Since the analytes are buried in the silica layer of TLC plate, it is important to develop a sampling method to transfer analytes from silica layer to mass analyzer. We develop a technique named ¡§TLCspray¡¨ to analyzing the chemical compounds deposited on TLC plate. The concept of TLCspray is similar to the direct electrospray probe (DEP) which has been described by our group: droplets deposed on a metal coated rod can producing ESI plume while applying high voltage. The TLC plate used for TLCspray is cut into stripes with sharp end. Samples are applied on the TLC stripes, than mount onto a holder which fix TLC stripes in front of mass inlet. The mobile phase flow through sample spot, and carry the analyte to the edge of TLC stripe where the fine ESI plume is produced in the effect of electric field. Complex matrixes such as red blood cell (RBC) and proteins are stop on the original position by the silica layer which acts as filter to clean up the unwanted interferences in biological samples. Zolpidem, a drug used to treat insomnia, is chose to demonstrate the sensitivity of TLCspray. The LOQ of zolpidem is 50 ng/mL, and linear dynamic range is 50-1000 ng/mL in human blood. The analysis process of TLCspray for human blood can be finished within 4 minutes. Comparing to other sample pretreatment prior to mass analysis for biological samples, TLCspray can reduce the time needed for clean up the samples without loss the sensitivity.

The analytical utility of gold nanoparticles (AuNPs) for laser desorption/ionization mass spectrometry (LDI-MS) is examined here. An evaluation of the parameters that affect desorption/ionization show that careful treatments of AuNPs is needed, as subtle changes in the solution environment can result in subsequent changes in the mass spectra. A thorough evaluation of the parameters that affect desorption/ionization of peptides is presented here, and these parameters include: (i) AuNP-to-analyte ratio, (ii) AuNP size, (iii) solvent, (iv) AuNP surface composition, (v) pH and buffer effects, (vi) amino acid sequence, and (vii) additives such as fructose or glycerol. Specifically, controlling the AuNP-to-analyte ratio, pH, peptide composition, and AuNP size are important parameters for ionization. Additionally, effects of passivating the AuNP surface with halides or oxyanions was investigated. The presence of NaF, NaCl, NaBr, and NH4X (X = F, Cl, Br, I) were shown to not significantly affect analyte ion abundances, whereas addition of NaI strongly suppressed analyte ion yields. Further physical characterization of the NPs showed that etching had occurred, which suggests that the surface chemistry of the NPs is important for desorption/ionization. Throughout these investigations, questions remain as to what the internal energies of peptides are after the desorption/ionization event, and how energy is deposited. Peptide ion fragmentation is examined under different solution conditions to evaluate the relative internal energies of peptides, and the fragmentation pattern examined for insight into fragmentation mechanisms. The data suggest that radical species are important for fragmentation of peptides when using AuNPs. However, it is likely that multiple processes are actually directing the fragmentation. Finally, based on the data presented in this dissertation, a thermal desorption mechanism of pre-formed ions is proposed. This fundamental research is intended to lay foundations for optimizing the use of nanoparticles in routine LDI-MS analysis as well as giving insight into nanoparticle ionization mechanisms. Since very little work has been done in this area, this dissertation investigates, in detail, many of the subtle characteristics that affect desorption/ionization of biomolecules when using NPs.

Peptide synthesis and metal doping, combined with mass spectrometric and ion mobility spectrometric techniques, have provided a picture of the fragmentation behavior of a large field of homologous peptide ions, represented as XVGVAZG, where the X amino acid is either arginine, histidine, lysine, aspartic acid or tryptophan and the Z amino acid is proline, glycine, serine, or histidine. These homologous peptide ions have been carefully selected to probe the effects of charge site location and secondary interactions upon the fragmentation chemistry of peptides. Peptides were synthesized on solid support, doped with appropriate metal salts to attach Li+, Na+, K+, Cu+ and Ag+ , and then examined using ion mobility spectrometry, and tandem mass spectrometry, both high energy collision induced dissociation (CID) and photodissociation using 193- nm laser light. Molecular dynamics calculations enabled me to derive candidate structures for these ions that agree with the ion mobility data for the ions. The fragmentation chemistry and structure selection of the first group of peptides, those that contain a proline residue, indicate that the presence of high proton and high metal ion affinity residues at the N-terminal position of the peptide direct the fragmentation of the highly charge-solvated ions according to a charge site directed mechanism. Further examples of charge-solvated structures and charge-directed fragmentation are shown for peptides where the sixth amino acid residue has been replaced with glycine or serine, eliminating the influence of the proline residue in the sixth position. Photodissociation of the peptides indicates that the position of valine residues along the peptide backbone influences the types of abundant fragment ions observed and ai and dai ions are observed exclusively at the site of valine residues. This observation continued, even when the position of the valine residues were altered by synthesis, leading me to the conclusion that the fragmentation of these peptides. The study was expanded to include significantly more complex peptides, those containing second high proton and high metal ion affinity residues, and though the data are complex, the influence of charge solvation in those systems is strong as well, according to my analysis of the candidate structures obtained and the types of fragment ions observed.