Prefractionation using microscale solution IEF.

Proteomics often involves systematic analyses of proteomes that are constantly changing in response to changes in the environment of the cell, tissue, or organism being analyzed. Due to limitations of all current protein profiling methods, powerful, reliable proteome prefractionation methods prior to two-dimensional electrophoresis (2DE) gels or alternative non-2DE gel methods are needed for in-depth quantitative comparisons of the complex proteomes typically encountered with samples from higher eukaryotes. The microscale solution isoelectrofocusing (MicroSol IEF) fractionation method is capable of reproducibly dividing complex proteomes into as many as seven well-resolved fractions based on the proteins' pIs on a small volume scale ( approximately 0.65mL/fraction). When MicroSol IEF is combined with narrow pH range 2DE gels or with alternative downstream analysis methods, it can substantially increase the detection dynamic range and the total number of proteins that can be quantitatively compared. Although MicroSol IEF is reasonably reproducible, subtle variations can occur in different separations similar to the minor variations often seen in most separations of proteins. Therefore, for reliable quantitative comparisons the samples to be compared should be differentially labeled with either Cy dyes or stable isotope labels prior to mixing and separation in a single MicroSol IEF run. Larger numbers of samples can be compared across many MicroSol IEF separations by using a differentially labeled internal standard composed of equal aliquots of all samples to be compared.

Higher dimensional (Hi-D) separation strategies dramatically improve the potential for cancer biomarker detection in serum and plasma.

The plasma proteome has a wide dynamic range of protein concentrations and is dominated by a few highly abundant proteins. Discovery of novel cancer biomarkers using proteomics is particularly challenging because specific biomarkers are expected to be low abundance proteins with normal blood concentrations of low nanograms per milliliter or less. Conventional, one- and two-dimensional proteomic methods including 2D PAGE, 2D DIGE, LC-MS/MS, and LC/LC-MS/MS do not have the capacity to consistently detect many proteins in this range. In contrast, new higher dimensional (Hi-D) separation strategies, utilizing more than two dimensions of fractionation, can profile the low abundance proteome.

Proteome analysis of Halobacterium salinarum and characterization of proteins related to the degradation of isopropyl alcohol.

We reported in a previous study that proteomic approach, coupled with genomic techniques, could be used to screen and develop multiple candidates for halophilic enzymes from Halobacterium salinarum. In order to evaluate the biodegradation of isopropyl alcohol (IPA) by H. salinarum, the amounts of residual IPA and acetone generated in the growth media were determined using a gas chromatography-flame ionization detector (GC-FID). The protein expression profiles of cells which had been cultured with IPA were obtained with the two-dimensional gel electrophoresis. Proteins evidencing different expression levels in the presence of 0.5% IPA were identified by electrospray ionization-quadruple-time of flight (ESI-Q-TOF) mass spectrometry. We found 12 proteins which were down-regulated, and another 12 proteins which were up-regulated, in the presence of 0.5% IPA and we further identified 17 proteins among them using ESI-TOF MS/MS. Among these identified proteins, we selected glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for further characterization as a halophilic enzyme. We have demonstrated for the first time that H. salinarum possesses the ability to degrade IPA and GAPDH was both stable and active at high salt concentrations, with maximum activity occurring at 1 M NaCl, although the optimal salt concentration with regard to the growth of H. salinarum is 4.3 M.

Protein detection in gels without fixation.

A number of alternative methods are described for detecting proteins in polyacrylamide gels that do not require fixation of the protein either prior to staining or in conjunction with staining. The primary advantage of avoiding fixation is that this makes it easier to remove proteins of interest from the gels for subsequent analysis. In general, the sensitivity of protein detection methods that avoid fixation is lower than for detection methods using fixation. For any given method, sensitivity is dependent on the volume of the protein band within the gel; hence, sensitivity is highest for sharp, narrow bands. Techniques described in this unit include protocols for protein detection in gels by SDS precipitation, preparation of contact blots, staining with imidazole-zinc, and use of the fluorescent labels IAEDANS and fluorescamine. Several additional methods, including the use of tryptophan fluorescence, guide strips, and minimal protein staining, are discussed in the Commentary.

Halophile aldehyde dehydrogenase from Halobacterium salinarum.

Halobacterium salinarum is a member of the halophilic archaea. In the present study, H. salinarum was cultured at various NaCl concentrations (3.5, 4.3, and 6.0 M NaCl), and its proteome was determined and identificated via proteomics technique. We detected 14 proteins which were significantly down-regulated in 3.5 M and/or 6 M NaCl. Among the identified protein spots, aldehyde dehydrogenase (ALDH) was selected for evaluation with regard to its potential applications in industry. The most effective metabolism function exhibited by ALDH is the oxidation of aldehydes to carboxylic acids. The ALDH gene from H. salinarum (1.5 kb fragment) was amplified by PCR and cloned into the E. coli strain, BL21 (DE3), with the pGEX-KG vector. We subsequently analyzed the enzyme activity of the recombinant ALDH (54 kDa) at a variety of salt concentrations. The purified recombinant ALDH from H. salinarum exhibited the most pronounced activity at 1 M NaCl. Therefore, the ALDH from H.salinarum is a halophilic enzyme, and may prove useful for applications in hypersaline environments.

Plasma protein level changes in waste incineration workers exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a chemical compound which is known to induce severe reproductive and developmental problems, immune system damage, and interference with regulatory hormones. To characterize changes in the expression of plasma proteins caused by exposure to TCDD, we analyzed plasma samples from workers at municipal incinerators using two-dimensional gel electrophoresis (2-DE). Proteins exhibiting differences in expression were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF mass spectrometry. One newly expressed protein was identified as the adrenomedulin binding protein (AMBP). Seven overexpressed proteins were identified in this study, and the most overexpressed protein was identified as alpha-fetoprotein (AFP). In addition, we cultured HepG2 cells in the presence of TCDD, to determine the effects of TCDD on the AFP and albumin expression in mRNA and protein levels, via RT-PCR and Western blotting, respectively. TCDD treatment resulted in an increase in the mRNA and protein expression levels of AFP, but reduced albumin expression. According to our results, exposure to TCDD may induce liver disease or cancer, and the proteins identified in this study could help reveal the mechanisms underlying TCDD toxicity.

Proteomic characterization of rat liver exposed to 2,3,7,8-tetrachlorobenzo-p-dioxin.

Dioxins are a class of polyhalogenated aromatic hydrocarbons that induce a wide spectrum of toxic responses in experimental animals. In this study, 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) was exposed to two SD rat groups; one group for short-term exposure at a single dose of 1, 10, 20 and 50 mug/kg body weight (group 1) and the other for long-term exposure at daily and-low dose of 0.01, 0.1, 1 and 2.5 microg/kg body weight (group 2) for a month. Two-dimensional electrophoresis (2-DE) was utilized to resolve the protein profile of rat liver exposed to TCDD at different doses. In the analysis of 2-DE of the group 1, two new-expressed spots and seven volume-increased spots were detected and identified by ESI-Q-TOF MS/MS; especially, proteasome subunit beta type 3 was increased in all doses. In addition, in the group 2, six volume-increased spots were screened; particularly, histidine triad nucleotide binding protein was increased in both 0.1 microg/kg dose and 1 microg/kg dose. The identified proteins were confirmed using Western blot. Among the identified proteins, apolipoprotein A-IV may protect lipid peroxidation and atherosclerosis induced by TCDD exposure and the expression level of phosphoglycerate mutase increases due to hyperthyroidism induced by TCDD exposure.

An efficient proteomics based strategy for the functional characterization of a novel halophilic enzyme from Halobacterium salinarum.

The extremely halophilic archaeon, Halobacterium salinarum grows in environments containing over 25% NaCl. The enzymes of this organism have thus been adapted to be active and stable in hypersaline conditions, which makes them strong candidates as robust industrial enzymes. In this study, the proteomics approach was applied to screen novel halophilic enzymes. We focused initially on proteins that are differentially expressed under different salt concentrations in culture media. After two-dimensional gel electrophoresis over a pH 3.5-4.5 range, 29 differentially expressed protein spots were identified by tandem mass spectrometry and six of these had no similarity to preexisting genes of known function. To predict the function of them, we used various bioinformatic methods. Among other proteins, we selected Vng0487h, which showed a high similarity to acetyltransferases. As a step toward assaying the enzymatic activity of this protein, we cloned the Vng0487h gene of H. salinarum and expressed and purified the recombinant protein with a glutathione-S-transferase (GST) tag in Escherichia coli. Using a GST-pulldown assay, a protein fragment derived from E. coli could interact with recombinant Vng0487h, and was identified to be the ribosomal protein L3. This protein showed high sequence homology with ribosomal protein L7/12 from E. coli and ribosomal protein L13p from H. salinarum. This suggests that Vng0487h acetylates a subunit of ribosomal protein, possibly L13p, in H. salinarum. During the present study, an efficient procedure was established to screen novel halophilic enzymes, and to predict and assess their functions.

Proteomics of Halophilic archaea.

Halophilic archaea is a member of the Halobacteriacea family, the only family in the Halobacteriales order. Most Halophilic archaea require 1.5M NaCl both to grow and retain the structural integrity of the cells. The proteins of these organisms have thus been adapted to be active and stable in the hypersaline condition. Consequently, the unique properties of these biocatalysts have resulted in several novel applications in industrial processes. Halophilic archaea are also to be useful for bioremediation of hypersaline environment. Proteome data have expended enormously with the significant advance recently achieved in two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The whole genome sequencing of Halobacterium species NRC-1 was completed and this would also provide tremendous help to analyze the protein mass data from the similar strain Halobacterium salinarum. Proteomics coupled with genomic databases now has become a basic tool to understand or identify the function of genes and proteins. In addition, the bioinformatics approach will facilitate to predict the function of novel proteins of Halophilic archaea. This review will discuss current proteome study of Halophilic archaea and introduce the efficient procedures for screening, predicting, and confirming the function of novel halophilic enzymes.

Differential expression of proteins in rat plasma exposed to benzene.

Benzene, a ubiquitous environmental contaminant, is an important solvent in the chemical industry and is also known as a constituent of petroleum. It has been reported that benzene is associated with hematotoxicity including leukemia in humans and cancer in laboratory animals. To study protein expression alterations in rat plasma exposed to benzene, rats were exposed to levels of 1, 10, 100 ppm benzine for 6 h/day and 5 d/week for 2 or 6 weeks. Two-dimensional gel electrophoresis of rat plasma was carried out, and approximately 1000 protein spots were detected on the gels. The 11 spots which showed significantly different expression were selected and identified with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. Analyzing the targeted 11 spots, there was no correlation between the 2 and 6 weeks benzene-inhaled groups on up-regulated proteins (zinc finger protein, and tristetraprolin) and on down-regulated proteins (cAMP-regulated guanine nucleotide exchange factor II, protein kinase and unknown protein). The overexpressed proteins (inhibitor of kappaB-like protein, GTP-binding protein rab14, T-cell receptor alpha chain, and somatostatin transactivating factor-1) were detected only in groups inhaling benzene for 6 weeks. Among them the expression level of T-cell receptor alpha chain was confirmed by Western blot.

Protein biomarkers in the plasma of workers occupationally exposed to polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) form a chemical family containing several hundred compounds, including benzo[a]pyrene and pyrene. They are usually produced by the incomplete burning of coal, oil, gas, garbage, or other organic substances like tobacco or charbroiled meat. Exposure to PAH causes tumors, primarily in the lung, the bladder, and the skin. To investigate the differentially expressed proteins resultant from PAH exposure, the protein expression in human plasma was analyzed using two-dimensional electrophoresis (2-DE). The plasma exposed to PAH was obtained from 48 waste gas pollution measurers working at an automobile emission inspection center. The 1-hydroxypryene (1-OHP) level, which is the urinary PAH metabolite used for evaluation of PAH exposure, was 0.28 micromol/mol creatinine in PAH exposure groups, and 0.078 micromol/mol creatinine in unexposed groups (control, n = 33). A protein upregulated by PAH (putative capacitative calcium entry channel) and five overexpressed proteins (two fibrinogen gamma-A chain precursors, a hemopexin precursor, an albumin precursor, and T-cell receptor beta chain C region) were identified with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and confirmed with tandem MS (MS/MS) and Western blotting. The putative capacitative calcium entry channel was partially validated with a laboratory made antibody of a representative peptide fragment in PAH-exposed human plasma samples.

Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum.

Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCl). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.

Identification of a novel protein D3UPCA from Halobacterium salinarum and prediction of its function.

Halobacterium salinarum is an extremely halophilic archaea, which is able to live in highly saline environments. In a recent study, several halophilic archaea were found to have the ability to biodegrade organic hydrocarbon pollutants, but protein information regarding hydrocarbon degradation and tolerance in halophilic archaea has been relatively rare. In this study, the protein expression profile of H. salinarum cultured under different diesel concentrations (0, 2 and 4%) was investigated by two-dimensional gel electrophoresis. Proteins which increased their expression levels in diesel media were identified using matrix-assisted laser desorption/ionization-time of flight and electrospray ionization-tandem mass spectrometry analysis. Among these, a protein spot (named D3UPCA) which was up-regulated about nine-fold and found to have COG3388, an uncharacterized protein conserved in archaea, was selected in order to further characterize its functions. The D3UPCA coding gene (named d3upca) was cloned and expressed in Escherichia coli, and purified by the glutathione-S-transferase-fusion method. The function of the protein was estimated using various bioinformatics tools and was predicted to be related to the regulation of transcription and/or translation of genes needed to tolerate stresses associated within the presence of diesel oil.

Analysis of differentially regulated proteins in TM4 cells treated with bisphenol A.

BPA, bisphenol A, a monomer of epoxy resins and polycarbonate plastic, is used in many consumer products including the plastic linings of cans for food and babies' bottles. BPA has been reported to cause reproductive toxicity and affects cells in rats and mice at high doses. In this study, the effect of BPA on protein expression in TM4 cells (a mouse Sertoli cell line) known to play an essential role in Spermatogenesis was investigated by two-dimensional electrophoresis (2-DE). After 16 h exposure to 50, 100, 150, 200, and 250 microM of BPA, the viability of TM4 cells decreased to about 90, 85, 78, 55, and 30% of control respectively. Approximately 800 protein spots in TM4 cells were analyzed by 2-DE with pH 4-7 linear immobilized pH gradient (IPG) Dry Strip, and 11 proteins which showed significantly different expression levels were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Among these, HSP 27 and placental calcium binding protein may be proteins differentially expressed by BPA exposure.

Proteomic analysis of plasma proteins of workers exposed to benzene.

In this study, we analyzed the proteins in plasma of workers exposed to benzene by two-dimensional gel electrophoresis, in the hope of finding a specific protein suitable for the biomonitoring of benzene exposure. Comet assays were also carried out to evaluate lymphocytes DNA damage. Fifty workers from a printing company and 38 matched unexposed healthy subjects were enrolled in the study. DNA damage was found to be significantly higher in the exposed workers than in the controls. The tail moments of the two groups were 2.07 +/- 0.35 and 1.48 +/- 0.41, respectively (P < 0.0001). The mean values of trans, trans-muconic acid (t,t-MA) in workers exposed to benzene and in unexposed subjects were 1.011 +/- 0.249 and 0.026 +/- 0.028 mg/g creatinine, respectively. Protein profiles were significantly different (P < 0.05) in the two groups, as identified by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry and confirmed by Western blot. T cell receptor beta chain (TCR beta), FK506-binding protein (FKBP51) and matrix metalloproteinase-13 (MMP13) were found to be up-regulated in the benzene-exposed workers. In addition, the correlation between TCR beta and the tail moments of lymphocytes was statistically significant (r-value, 0.428). We conclude that TCR beta in plasma could be used for the early detection of exposure to benzene.

Analysis of proteins expressed in rat plasma exposed to dioxin using 2-dimensional gel electrophoresis.

Dioxins are a class of polyhalogenated aromatic hydrocarbons that induces a wide spectrum of toxic responses in animals. In this study, two groups of Sprague-Dawley rats were exposed to 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD); one group received short-term exposure at a single dose of 1, 10, 20 or 50 microg/kg body weight and the other received long-term exposure to a daily low dose of 0.01, 0.1, 1 or 2.5 microg/kg body weight for one month. Two-dimensional electrophoresis was utilized to resolve the protein profile of rat plasma exposed to TCDD at different doses. One novel and three volume-increased spots were identified and characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray-ionization on quadropole-TOF2 mass spectrometry. The novel protein was identified as plasma glutathione peroxidase precursor and the volume-increased proteins were cytokeratin 8 polypeptide, Ig lambda-1 chain C region and Ig lambda-2 chain C region. These proteins may be used as biomarkers to diagnose dioxin exposure and may help in understanding the toxic effects of dioxins.

Monitoring protein expression by proteomics: human plasma exposed to benzene.

Low levels and long term exposure to benzene is associated with hematotoxicity including aplastic anemia, acute myelogenous leukemia, and lymphoma. Current biomonitoring methods such as urinary phenol, S-phenylmercapturic acid, and trans-trans muconic acid were found to be unreliable as analytical methods to detect benzene exposure. Therefore, to search for a specific protein for biomonitoring benzene exposure, we investigated plasma proteins from workers (n = 50) at a printing company who were exposed to benzene, by two-dimensional gel electrophoresis. The protein profiles are significantly different (p < 0.05) between benzene exposed and unexposed groups, as identified by matrix-assisted laser desorption ionization/time of flight mass spectrometry and confirmed by Western blot analyses. T cell receptor beta chain (TCR beta), FK506-binding protein, and matrix metalloproteinase-13 were expressed only in benzene exposed workers. In addition, interleukin-4 receptor alpha chain and T cell surface glycoprotein CD1b precursor were found to be up-regulated in the plasma of benzene exposed workers. When we treated Jurkat cells with benzene (10 microM-10 mM), TCR beta expression was increased in the membrane more than 6-9-fold compared to untreated cells. In addition, the amount of TCR beta released into the culture media, at benzene concentrations greater than 50 microM, increased up to 10 mM. Therefore, TCR beta levels in plasma could be used as a biomarker and a possible therapeutic target for benzene exposure.

Development of an effective sample preparation method for the proteome analysis of body fluids using 2-D gel electrophoresis.

A sample preparation is still the most critical step in two-dimensional electrophoresis (2-DE) and should be optimized for each type of sample. In this study, a protein extraction method from body fluids was developed using a combined centrifugal filter device and a sample treating buffer. When plasma, amniotic fluid, urine, and tear were tested with this method, the recovery of protein reached almost 90% and high-quality separation of 2-DE gel was obtained.