A proteomic approach based on multiple parallel separation for the unambiguous identification of an antibody cognate antigen.

Autoantibodies obtained from cancer patients have been identified as useful tools for cancer diagnostics, prognostics, and as potential targets for immunotherapy. Serological proteome analysis in combination with 2-DE is a classic strategy for identification of tumor-associated antigens in the serum of cancer patients. However, serological proteome analysis cannot always indicate the true antigen out of a complex proteome identified from a single protein spot because the most abundant protein is not always the most antigenic. To address this problem, we utilized multiple parallel separation (MPS) for proteome separation. The common identities present in the fractions obtained using different separation methods were regarded as the true antigens. The merit of our MPS technique was validated using anti-ARPC2 and anti-PTEN antibodies. Next, we applied the MPS technique for the identification of glycyl-tRNA synthetase as the cognate antigen for an autoantibody that was overexpressed in the plasma of breast cancer patients. These results reveal that MPS can unambiguously identify an antibody cognate antigen by reducing false-positives. Therefore, MPS could be used for the characterization of diagnostic antibodies raised in laboratory animals as well as autoantibodies isolated from diseased patients.

Evaluation of toxicological monitoring markers using proteomic analysis in rats exposed to formaldehyde.

Formaldehyde (FA) is known as a low molecule weight organic compound and one of major components that causes sick building syndrome (SBS), and it has been reported that FA has cytotoxic, hemotoxic, immunotoxic, and genotoxic properties. The International Agency for Research on Cancer (IARC) has characterized FA as a carcinogen. In this study, we investigated the effects of FA on rat plasma proteins by using proteomic approach. Rats were exposed to three different concentrations of FA (0, 5, 10 ppm) for 2 weeks at 6 hours/day and 5 days/week in an inhalation chamber. Malondialdehyde (MDA) assay and carbonyl spectrometric assay were conducted to determine lipid peroxidation and protein oxidation levels and Comet assays were used for genotoxicity evaluation. Level of MDA, carbonyl insertion and DNA damage in plasma, livers, and in the lymphocytes of rats exposed to FA were found to be dose dependently increased. Proteomic analysis using three different pI ranges (3.5-5.6, 5.3-6.9, 6-9) and large size two-dimensional gel electrophoresis (2-DE) showed the presence of 3491 protein spots. A total of 32 (19 up- and 13 down-regulated) proteins were identified as biomarkers of FA, all showed dose dependent expressions in the plasma of rats exposed to FA and of these, 27 protein spots were identified by MALDI-TOF/MS. Several differentiated protein groups were found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up- or down-regulated. Among these, the identities of SNAP 23, apolipoprotein A-1 and E, clusterin, kinesin, and fibrinogen gamma were confirmed by Western blot assay, and apo E was further analyzed by using 2-DE immunoblot assays to determine isoform patterns. Two cytokine including IL4 and INF-gamma were measured in plasma with respect to fibrinogen gamma changes. In summary, cytotoxicity, and genotoxicity assays, namely MDA lipid peroxidation assay, the carbonyl protein oxidation assay, and Comet genotoxic assay showed that these effects increased on increasing FA levels. Proteomic analysis with three different pI ranges and long size 2-DE gel electrophoresis showed that 32 protein spots were up-or down-regulated. Of these 32 proteins, 7 proteins were confirmed by western blot assay. They could be potential biomarkers for human diseases associated with FA exposure.

Oxidative damages in the DNA, lipids, and proteins of rats exposed to isofluranes and alcohols.

DNA damage, lipid peroxidation and protein oxidation were evaluated in rats exposed to a 1% isoflurane atmosphere with or without alcohol administration (administrated by gastric intubation at 4 g/kg body weight as a 50% solution). Single cell gel electrophoresis assays were performed in order to evaluate DNA damage occurring in the lymphocytes, spleen, bone marrow, brain, livers and lung of rats exposed to 1% isoflurane for 30 or 60 min with/without ethanol. Levels of malondialdehydes (MDA), a metabolite of lipid peroxidation, were determined in plasma and tissues. Carbonyl contents were also analyzed to determine levels of protein oxidation in plasma and tissues. Levels of DNA damage in lymphocytes, bone marrow, and the organ tissues of rats exposed to isoflurane were found to increase time dependently, and alcohol increased DNA damage. Lipid peroxidation and protein oxidation results showed patterns that differed from those of DNA damage. Levels of MDA in plasma, bone marrow, spleen, and the livers of rats exposed to isoflurane with/without ethanol were found to be time dependently increased, but this was not observed in the brain or lung. However, protein oxidation levels were significantly increased in the plasma, brains, and lungs of rats exposed to isoflurane, and exposure to isoflurane and alcohol, significantly increased these levels in plasma and brain. The present study demonstrates that isoflurane exposure results in significant DNA damage in rat lymphocytes, bone marrow, spleen, brain, livers, and lung. Moreover, alcohol was found to be as a strong inducer of DNA damage, lipid peroxidation and protein oxidation. However, no evidence in association between DNA damage, lipid peroxidation and protein oxidation was found. Regarding the effects of isoflurane and alcohol on oxidative damages, single strand DNA damages may be a useful biomarkers and blood cells and plasma appear to be more sensitive targets to oxidative damage than other tissues.

Effects of benzo(a)pyrene on protein expression in Jurkat T-cells.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants of air, water and soil, and are produced by the incomplete combustion of organic materials. The International Agency for Research on Cancer has characterized PAHs as carcinogens. In this study, we investigated the effects of benzo(a)pyrene (B(a)P), which is the most carcinogenic member of the PAHs, on Jurkat cell protein by proteomic analysis. Jurkat cells were treated with various concentrations of B(a)P (0, 2.5, 5, 10, 20 or 40 microM) for 24 or 48 h and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and lactate dehydrogenase assays were carried out to determine cytotoxicity and a Comet assay was used to determinate genotoxicity. The cytotoxicity assays showed that 2.5 microM of B(a)P was the maximal concentration that did not cause any toxicity, but nevertheless, at this level B(a)P produced significant DNA damage in Jurkat cells at 48 h. Proteomic analysis using three different pI ranges and large two-dimensional gel electrophoresis showed 3427 protein spots. A total of 46 (13 up- and 33 down-regulated) proteins were identified as biomarkers of B(a)P and showed dose-dependent expressions in Jurkat T-cell line exposed to B(a)P. Of these, 27 protein spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Two functionally differentiated protein groups were found. The protein group involving apoptosis and tumor suppression were found to be up-regulated, and B(a)P down-regulated enzyme was involved in energy metabolism, DNA synthesis and in cell structure and motility.