ABSTRACT
Removal of high abundance proteins is widely used in sample processing for proteomics studies of blood plasma. Immunoaffinity (IA) depletion is currently the most specific method for performing this step. Historically, IA depletion matrices have been designed to be used with inorganic buffers. However, the presence of salts in depleted samples presents a particular problem, and these must be removed in order to make samples compatible with post-depletion processing. Desalting (dialysis, ultrafiltration, size-exclusion, etc.) usually diminishes sample integrity due to labware associated losses. Moreover, these steps require additional labor, increasing the processing time and cost of analysis. In order to avoid these problems, we have developed an IA method using a volatile buffer that can be removed from depleted samples by lyophilization. This method allows the execution of reproducible and efficient depletion of blood plasma in a semi-automated manner.
Subject(s)
Blood Proteins/isolation & purification , Buffers , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Freeze Drying , Humans , Plasma , Reproducibility of Results , UltrafiltrationABSTRACT
Two receptors [estrogen receptor (ER)alpha and ERbeta] mediate the manifold effects of estrogens throughout the body. Although a clear role has been established for ERalpha in the classical effects of estrogen activity, the physiological role of ERbeta is less well understood. A small-molecule ERbeta selective agonist, ERB-041, has potent antiinflammatory activity in the Lewis rat model of adjuvant-induced arthritis. To characterize the response of target organs and pathways responsible for this antiinflammatory effect, mRNA expression profiling of the spleen, lymph node, and liver was performed, in conjunction with a global analysis of the plasma proteome. We find that the expression of a large number of genes and proteins are altered in the disease model and the majority of these are partially or fully reversed by ERB-041 treatment. Regulated pathways include the acute-phase response, eicosanoid synthesis, fatty acid metabolism, and iron metabolism. In addition, many of the regulated genes and proteins are known to be dysregulated in human rheumatoid arthritis, providing further evidence that the manifestations of the Lewis rat adjuvant-induced arthritis model bear similarity to the human disease.
Subject(s)
Arthritis, Experimental/metabolism , Arthritis, Rheumatoid/metabolism , Blood Proteins/metabolism , Estrogen Receptor beta/agonists , Oxazoles/therapeutic use , RNA, Messenger/metabolism , Animals , Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/drug therapy , Arthritis, Rheumatoid/drug therapy , Biomarkers/metabolism , Gene Expression Profiling , Liver/metabolism , Lymph Nodes/metabolism , Male , Organ Specificity , Random Allocation , Rats , Rats, Inbred Lew , Spleen/metabolismABSTRACT
Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of beta-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.