We have undertaken the following projects:? Alzheimer's disease: Mouse MAb 6E10 is a commercially available Ab used extensively in Alzheimers research. We have successfully identified 95% of the light chain and 82% of the heavy chain sequences of this Ab, with five of the six CDRs completely elucidated. We identified pronounced sequence microheterogeneities for the light chain CDR2 region, indicating that changes at the protein level derived from somatic hypermutation of the Ig VL genes in mature B-cells might contribute to unexpected structural diversity. Also, major glycoforms at the conserved heavy chain glycosylation site were determined to be core-fucoslylated, biantennary, complex glycans. We are identifying the sequence of anti--amyloid antibodies and polyclonal A-autoantibodies that have been isolated from human serum. The discovery of A-autoantibodies implies that AD is an autoimmune disorder. The characterization of these autoantibodies may prove useful in designing synthetic autoantibodies to enhance the bodys recognition and clearing of circulating A-peptide. The ratios of IgG1-4 were determined for the polyclonal Abs in this study and levels of IgG3,4 were found to be higher than found in total serum IgG. Additionally, levels of G2F glycans were higher in IgG2 than in controls and the level of bisecting GlcNAc was found to be lower for the A-autoantibody IgG1. These differences may point to changes in the autoantibodies that may reduce their ability to protect against plaque formation or may indicate additional physiological roles of the different A-IgG autoantibody subclasses. ? 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. The applicability of this technique is being utilized to determine sites of phosphorylation in CD34. A total of five sites of phosphorylation were identified from the in vitro kinase treatment, S313, S337/338, S343, T365/S366, and S371. In vivo phosphorylation sites on full-length mouse CD34 from HEK293F cells were identified. at S343 and at S337/338. Even though phosphorylation at these two sites was observed in the in vitro kinase experiments, they are not PKC sites; thereby, implicating that kinases other than PKC are potentially involved in CD34 signaling events.? Lipopolysaccharide (LPS), a glycolipid component of the outer membrane of Gram-negative bacteria, is a potent initiator of the inflammatory response in macrophages and other cells. In macrophages, LPS signals through lipid rafts by selectively recruiting signaling proteins to this subcellular compartment. A SILAC (Stable Isotope Labeling in Cell Culture) based quantitative (LC-MS/MS) proteomics approach and a label-free proteomics approach have been applied to define the lipid raft proteome in: macrophages at rest; during early (5 min LPS treatment); and late (30 min LPS treatment). The combined proteomics approaches enabled global profiling of the rafteome and identification of LPS responsive proteins in lipid rafts. Survey of the proteomics data showed a significant presence of proteasome subunits in the lipid rafts with several being sensitive to LPS treatment. The role of proteasome in LPS-induced extracellular signal-regulated kinase (ERK) activation in the lipid raft was investigated and we found that both proteasomal activity in the lipid raft and overall lipid raft integrity are crucial to ERK activation by LPS. ? We continue to investigate the mechanisms of oxidative stress by spin trapping, e.g., with DMPO, oxidatively induced free-radicals followed by structural characterization using mass spectrometry. Our studies on spin-trapped protein have helped to identify individual steps involved in formation of the most stable radical and have identified how the radical site can be trans-ferred from one site to another in a protein and from one protein to another. Identification of immuno spin-trapped proteins has helped to identify physiologically important free radical processes. ? Cu,Zn-Superoxide Dismutase-driven Free Radical Modifications: Immuno-spin trapping and MS were used to study human and bovine Cu, Zn-superoxide dismutase (SOD1)/H2O2 driven reactions with human serum albumin and mouse brain supernatant. Both copper and carbonate radical anion-initiated oxidations were studied to clearly dif-ferentiate SOD1-induced oxidation via CO3 radical anionfrom that initiated by Cu release from the SOD1 active site. Six distinct gel bands from the reaction with the mouse brain super-natant were observed by Western analysis using the anti- DMPO polyclonal Ab. Proteins in each band were identified; thereby implicating these proteins as potential targets of CO3 radical anion. These results led to a new model of SOD1 driven, CO3radical anion-initiated protein oxidation. ? Radical formation in Carboxypeptidase B1 (CPB1): LPS induced systemic inflammation was used in C57BL6/J mice to identify potential protein radicals by spin-trapping with DMPO which may mediate the hosts uncontrolled inflammation in sepsis-like syndrome. Western analyses of mice spleens following LPS treatment indicated the formation of a DMPO-nitrone adduct. Mass spectrometric analyses of the cor-responding Coomassie-stained gel bands revealed carboxypeptidase B1 (CPB1) as one of the potential proteins. Follow-up immunoprecipation experiments confirmed CPB1 as an acute phase protein and suggest a significant role for xanthine oxidase and eNOS in CPB1 free radical formation and nitration which may have a role in the development of systemic inflammatory response syndrome. ? Procainamide (PA) induced myloperoxidase (MPO) radical: The hypothesis of this study was that PA oxidation by MPO/H2O2 would produce PA cation radicals which could lead to free-radical oxidation of MPO. Using Western analyses and electron spin reso-nance spectroscopy, a protein radical on MPO was detected following the reaction. In addition, protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60) were detected using the anti-DMPO Ab. Mass spectrometric analyses of the corresponding Coomassie band confirmed the identity of the protein as human MPO. Since a side effect of procainamide treatment is agranulocytosis, it is proposed that the MPO radical may also play a role in agranulocytosis.? Immuno-spin trapping: The Mason Lab has developed an immuno-spin trapping technique to provide increased specificity for detection of spin-trapped proteins. The sen-sitivity of this approach is lower than that usually attained by MS in the full scan mode. In collaboration with the Mason Lab, a method involving collection of LC fractions followed by ELISA detection was developed. The ELISA positive fractions were then concentrated prior to MS analyses. This approach has the advantage of improved selectivity and sensitivity
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