We have undertaken the following projects:? Identification of glycans on the hepatitis C virus E2 glycoprotein: ? The HCV envelope protein E2 is heavily glycosylated. Studying the glycan moieties attached to the envelope E2 glycoprotein is important because the N-linked glycans on E2 protein are involved in the interaction with some human neutralizing antibodies, and may also have a direct or indirect effect on protein folding. In the present study we report the mass spectrometric characterization of the glycan moieties attached to the E2 glycoprotein. The mass spectrometric analysis clearly identified the nature, composition and microheterogeneity of the sugars attached to the E2 glycopeptides. All 11 sites of glycosylation on E2 protein were characterized, and the majority of these sites proved to be occupied by high mannose glycans. However, complex type oligosaccharides, which have not been previously identified, were exclusively observed at two N-linked sites and their identity and their heterogeneity were determined. (manuscript submitted)? ? Histone phosphorylation: ? In collaboration with the Archer Lab (LMC), we have investigated changes in the phosphorylation levels at specific sites on human and mouse linker histones as a function of treatment with glucocorticoids. In this study mouse 1471.1 cells and human U2OS cells were treated with dexamethasone and staurosporine. We quantitated phosphorylation levels at the protein level by ESIMS and identified phosphorylation changes at specific amino acids by tandem mass spectrometry. We found significant changes in phosphorylation levels of histones H1.3, H1.4, and H1.5 in mouse cells and H1.2 and 1.4 in the human cells. We identified three sites of phosphorylation on both mouse and human histone H1.4 that were responsive to these treatments. (manuscript ready for submission)? ? Improved methods for isolation and identification of phosphorylated peptidesproteins.? As a means of enriching for phosphorylated peptides, we hasve implemented immobilized titanium dioxide as a phosphopeptide enrichment technique for the MS identification of phosphorylation sites in proteins (manuscript in preparation). The applicability of this technique is being utilized to determine sites of phosphorylation in CD34 (R. Tennant collaboration) and tristetraprolin (P. Blackshear collaboration).? ? ? Top-Down Protein Characterization? Top-down protein characterization is based on obtaining sequence information from an intact protein. Fragmentation is commonly induced by sustained off-resonance irradiation collisional activation, electron capture dissociation, infra-red multiphoton dissociation, or a combination of these techniques. Because of the complexity of the spectra obtained, high resolution instrumentation, typically a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer, is used in the analysis. We have applied these techniques to two investigations: ? a) Penaeidins: Penaeidins are antimicrobial peptides from shrimp that are unique in that they are composed of two very different domains an N-terminal proline-rich domain (PRD) linked to the C-terminal cysteine-rich domain (CRD). We have used top-down proteomics (protein characterization at the intact protein level using Fourier Transform MS with electron capture dissociation and multiphoton ionization dissociation. Our results indicate that the CRD is compact and does not readily undergo fragmentation at the intact whole polypeptide level while fragmentation occurs readily within the PRD. (manuscript in press; Cuthbertson, BlackshearLN)? b) Human myoglobin spin-trapped free radical: In collaboration with the Mason Lab, we have investigated the structure of the DMPO spin-trapped myoglobin free radical by top down proteomics and compared the results with the bottom-up approach(proteolysis followed by peptide analysis). Although information was obtained about the location of the spin trap by top-down approach, it was not as specific as to the location as was the bottom-up approach.? ? We conclude from these experiments that, although the top-down approach has been reported to work with large polypeptides (ca. 12 kD and below), the richest fragmentation information is obtained from the N- and C-terminal regions with much less coverage of the middle of the protein. Thus, success with larger peptides is not at the level that it can be used to characterize the whole protein nor is it necessarily possible to locate sites of modifications. (Anal. Chem. in press)? ? Protein-DNA Interactions? Development of Protein-DNA Crosslinking techniques:? As part of our collaborative efforts with the DNA Repair and Nucleic Acid Enzymology Group at the NIEHS (S. Wilson, LSB) we have been working very closely with R. Prasad on designing and developing a crosslinkingenrichment approach for the MS analysis of protein-DNA crosslinks. This approach initially implemented biotinylated tags to the oligonucleotides for specific enrichment. To minimize the size of oligonucleotide remaining after crosslinking (as this can interfere with MS analyses), our was to engineer in a disulfide bond into the oligonucleotide sequence such that the disulfide bond could be reduced at a predetermined location; thereby, minimizing the size of oligonucleotide. Thus far, the disulfide-containing biotinylated oligonucleotide has been shown to efficiently crosslink with polymerase gamma. These reactions are currently being scaled-up for MS analyses.? Binding Partners of Polymerase Beta involved in Base Excision Repair: ? The DNA Repair and Nucleic Acid Enzymology Group at the NIEHS (S. Wilson, LSB) has extensively studied the mammalian base excision repair pathway. In particular, they have studied the role of DNA polymerase beta within this pathway. As part of our collaboration with the Wilson Lab (R. PrasadS. Wilson - LSB) to identify binding partner(s) of pol beta involved in base excision repair we identified HMGB1 protein as specifically interacting with the base excision repair intermediate (Mol. Cell, in press), We are currently collaborating with P. Kedar in the Wilson Lab to identify additional proteins of interest in the BER pathway.? ? Oxidative Stress? Although oxidative stress (state of high levels of free radicals) is thought to be involved in the pathogenesis of several diseases and aging, the mechanisms by which free radicals react is not well understood. We have been collaborating for a number of years with The Free Radical Metabolites Group at the NIEHS (R. Mason, LPC) to study the mechanisms associated with oxidative stress. In particular, we have been investigating the nature of radicals formed on several heme-containing proteins. Most recently, we have collaborated with the Mason laboratory on the development and optimization of the anti-DMPO antibody for co-immunoprecipitation of DMPO-containing proteins. These analyses are being performed on both in vivo and in vitro assays. We have identified the kinetic and thermodynamic products of free radical formation of human myoglobin (J. Am. Chem. Soc., in review).? ? ALS? We are iniating a collaboration with F. Kamel and B.A. Merrick to identify potential serum biomarkers for ALS.
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