Use Of Two-dimensional Gel Electrophoresis Research Articles

Deep Imaging: How Much of the Proteome Does Current Top-Down Technology Already Resolve?

Effective proteome analyses are based on interplay between resolution and detection. It had been claimed that resolution was the main factor limiting the use of two-dimensional gel electrophoresis. Improved protein detection now indicates that this is unlikely to be the case. Using a highly refined protocol, the rat brain proteome was extracted, resolved, and detected. In order to overcome the stain saturation threshold, high abundance protein species were excised from the gel following standard imaging. Gels were then imaged again using longer exposure times, enabling detection of lower abundance, less intensely stained protein species. This resulted in a significant enhancement in the detection of resolved proteins, and a slightly modified digestion protocol enabled effective identification by standard mass spectrometric methods. The data indicate that the resolution required for comprehensive proteome analyses is already available, can assess multiple samples in parallel, and preserve critical information concerning post-translational modifications. Further optimization of staining and detection methods promises additional improvements to this economical, widely accessible and effective top-down approach to proteome analysis.

Changes in the seminal plasma proteome of adolescents before and after varicocelectomy

To compare seminal plasma protein profiles before and after varicocele correction to assess if surgical intervention alters the protein profile. Prospective study. Academic research environment. Nineteen adolescent boys with varicocele grades II or III. Two semen samples were collected before bilateral subinguinal microsurgical varicocelectomy, and two semen samples were collected 3 months after surgery. Seminal plasma protein profiles were determined with the use of two-dimensional gel electrophoresis. Proteins were separated in 18-cm 3-10 pH strips and 10%-17.5% gradient gels. Gels were stained, scanned, and compared with the use of Imagemaster 2D platinum 7.0. Spots of interest were removed from gels, and protein digestion was performed with the use of trypsin. Digests were identified with the use of electrospray ionization-quadrupole/time-of-flight tandem mass spectrometry (ESI-QTOF MS/MS), and spectra were analyzed with the use of the Mascot software. Proteins uniquely or overexpressed in each period (before or after varicocelectomy). Nineteen spots were differentially expressed between pre- and postsurgery samples. Identified proteins were albumin, proteasome subunit alpha type 6, alpha-1-antitrypsin, fibronectin, CD177, prostatic acid phosphatase, specific prostatic antigen, alpha-2-antiplasmin, vitamin D-binding protein, gastricsin, clusterin, semenogelin-1, semenogelin-2, superoxide dismutase, protein-glutamine gamma glutamyltransferase-4, and prolactin-inducing protein. Varicocelectomy is associated with changes in the seminal plasma protein profile. Understanding specific pathways leading to male infertility may further assist physicians in demonstrating deviation from homeostasis in male infertility. In addition, it may be possible to observe if surgical intervention does indeed revert altered pathways toward a homeostatic state.

106P PROTEOMIC ANALYSIS SHOWED HEAT SHOCK 70 KDA PROTEIN AND 78 KDA GLUCOSE-REGULATED PROTEIN WERE UP-REGULATED IN HUMAN MALIGNANT PLEURAL MESOTHELIOMA CELLS

Malignant pleural mesothelioma (MPM) is a malignant neoplasm arising from mesothelial cells existing in pleura. It is difficult to identify a crucial symptom in patients with MPM, and there is no standard curative therapy for this malignancy. The incidence of MPM is increasing worldwide, and many patients can not survive more than one year after diagnosis. MPM is an asbestos-related disease, and it is believed that the incidence is closely related to the amount and time of exposure to asbestos. Since the latency period after exposure to asbestos is very long, the incidence may increase over the next two decades. Therefore, it is important to understand the biology of MPM, and to find a molecular target for its therapy. Three of human malignant pleural mesothelioma (MPM) cell lines (NCIH28, NCI-H2052, NCI-H2452) and a human pleural mesothelial cell line (MeT-5A) were analyzed by proteomics with the use of twodimensional gel electrophoresis (2-DE) and liquid chromatographytandem mass spectrometry (LC-MS/MS). From the results of 2-DE between MeT-5A and three MPM cell lines, 9 protein spots showed increased expression levels in MPM cells compared to MeT-5A. These spots were analyzed by LC-MS/MS analysis, and identified by a peptide sequence tag. Identified increased spots included 78 kDa glucose regulated protein (GRP78), heat shock 70 kDa protein (HSP70), protein disulfide isomerase A3, annexin A3, peroxiredoxin-2 and 14 kDa phosphohistidine phosphatase. Western blotting using specific antibodies against GRP78 and HSP70 confirmed the elevated expression level of GRP78 and HSP70 in MPM cell lines compared to MeT-5A cells. These results suggest that GRP78 and HSP70 expression is likely to be associated with the tumorigenesis of MPM, and they may be expectable as biomarkers or therapeutic targets for MPM. Disclosure: All authors have declared no conflicts of interest.

107P IMPACT OF PREINTERVENTIONAL PLASMA FIBRINOGEN AS A PROGNOSTIC MARKER IN MALIGNANT PLEURAL MESOTHELIOMA

Malignant pleural mesothelioma (MPM) is a malignant neoplasm arising from mesothelial cells existing in pleura. It is difficult to identify a crucial symptom in patients with MPM, and there is no standard curative therapy for this malignancy. The incidence of MPM is increasing worldwide, and many patients can not survive more than one year after diagnosis. MPM is an asbestos-related disease, and it is believed that the incidence is closely related to the amount and time of exposure to asbestos. Since the latency period after exposure to asbestos is very long, the incidence may increase over the next two decades. Therefore, it is important to understand the biology of MPM, and to find a molecular target for its therapy. Three of human malignant pleural mesothelioma (MPM) cell lines (NCIH28, NCI-H2052, NCI-H2452) and a human pleural mesothelial cell line (MeT-5A) were analyzed by proteomics with the use of twodimensional gel electrophoresis (2-DE) and liquid chromatographytandem mass spectrometry (LC-MS/MS). From the results of 2-DE between MeT-5A and three MPM cell lines, 9 protein spots showed increased expression levels in MPM cells compared to MeT-5A. These spots were analyzed by LC-MS/MS analysis, and identified by a peptide sequence tag. Identified increased spots included 78 kDa glucose regulated protein (GRP78), heat shock 70 kDa protein (HSP70), protein disulfide isomerase A3, annexin A3, peroxiredoxin-2 and 14 kDa phosphohistidine phosphatase. Western blotting using specific antibodies against GRP78 and HSP70 confirmed the elevated expression level of GRP78 and HSP70 in MPM cell lines compared to MeT-5A cells. These results suggest that GRP78 and HSP70 expression is likely to be associated with the tumorigenesis of MPM, and they may be expectable as biomarkers or therapeutic targets for MPM. Disclosure: All authors have declared no conflicts of interest.

PI-Based Separation of Tissue Proteins Realized with the Use of Two-Dimensional Gel Electrophoresis or Alternatively Capillary Isoelectric Focusing

A two-dimensional gel electrophoresis (2D-GE) and capillary isoelectric focusing (cIEF) were performed sub- sequently to analyze protein mixtures from laboratory animal tissues (liver, muscle, and mammary gland). Both tech- niques were compared and evaluated. It was found that cIEF can be a valuable alternative in comparison to 2D-GE. Espe- cially considering the goals associated with the determination of far the separated proteins, cIEF possesses perspective analytical advantages.

A proteomic analysis of the effect of growth hormone on mammary alveolar cell-T (MAC-T) cells in the presence of lactogenic hormones

The bovine mammary alveolar cell-T (MAC-T) cell line is able to uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin, and prolactin and is extensively used to study bovine mammary epithelial cell (MEC) function. Somatotropin, or growth hormone (GH), has been shown to increase milk protein synthesis both in vivo and in mammary cell models and to induce cytoskeletal rearrangement in a 3T3 fibroblast cell line and a Chinese hamster ovary cell line. To identify the nature of the effects of GH in MECs cultured with lactogenic hormones, changes in global protein expression were assessed in the MAC-T cell line with the use of two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry. Forty proteins were differentially expressed in response to GH (P < 0.05) and were related to metabolism, the cytoskeleton, protein folding, RNA and DNA processing, and oxidant stress. These widespread changes in protein expression are indicative of a global role of GH in overall cellular differentiation that may underlie the direct modulation of milk component synthesis in MEC models that have been described to date.

Influence of the malting parameters on the haze formation of beer after filtration

Malting changes the chemical and enzymatical composition of barley. During malting, enzymes are synthesized, cell walls (pentosans, proteins, etc.) degraded and starch becomes available for enzymatic attack. The progress of germination defines the final beer quality and processability in several aspects: mouthfeel, foam and haze formation (different proteins), processability (viscosity caused by certain substances, like β-glucan), fermentation progress (FAN, sugar content), etc. The objective of this research was to study the influence of different modified malt on turbidity in beer after filtration. This was achieved by analyzing selected malts at different germination stages and afterward studying their influence on the final beer composition, focusing on protein content and composition. Protein fractions were analyzed using a Lab-on-a-Chip technique, which separates the proteins—based on their molecular weight—by capillary electrophoresis. This analysis was supported by the use of two-dimensional gel electrophoresis (2D-PAGE). Additionally, common malt and beer analyses and turbidity and filterability measurements were performed. The protein composition could be followed from malt to beer with both the Lab-on-a-Chip technique and 2D-PAGE. No differences in protein composition could be seen in the final protein composition of the beer. However, it could be observed, with Lab-on-a-Chip technique, that high amounts of a protein fraction with a size of 25–28 kDa caused increased turbidity in the beer.

Proteome alterations of cortex and hippocampus tissues in mice subjected to vitamin A depletion

Vitamin A regulates the development and maintenance of the central nervous system. Studies of vitamin A depletion (VAD) and mutations of retinoid receptors in rodents have revealed a dysfunction of motor and cognitive abilities. However, the molecular mechanisms underlying these behavioral changes are not well understood. In this study, VAD mice were examined and abnormal motor behavior related to psychosis symptoms was found. With the use of two-dimensional gel electrophoresis (2-DE), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometric (MS) technologies, 44 and 23 altered protein spots were identified in the cortex and hippocampus, respectively, in VAD mice. By Western blot, the up-regulation of mitogen-activated protein kinase 1 (MAPK1) and proteasome subunit beta type 2 (PSMB2) in the cortex and that of dihydropyrimidinase-related protein 2 (DPYSL2) and PSMB2 in the hippocampus were observed in VAD mice. Bioinformatic analysis using DAVID revealed that altered proteins induced by VAD showed significant enrichment of (i) glycolysis, cytoskeleton, mitochondrion and glutamate metabolism in the cortex; and (ii) actin binding, dopamine receptor signaling and transmission of nerve impulse in the hippocampus. The up-regulations of DPYSL2, MAPK1 and PSMB2 may indicate the activated neuronal defensive mechanism in VAD brain regions, which may underlie the VAD-related psychosis behavior.

The star chart to Ta bladder cancer: An unsophisticated analysis of two-dimensional gel electrophoresis proteome maps

To explore the use of two-dimensional gel electrophoresis (2DE) for analysing the proteome of clinically relevant tissue samples such as biopsies from transurethral resections of the bladder (TURB), by generating a Ta proteome map, possibly identifying technical or biological artefacts, and searching for biological subgroups associated with clinical data. Biopsies from 23 patients were homogenized and the protein content was separated by 2DE. The gels were silver stained and scanned, and the resulting pictures were analysed for similarities in the spot pattern. A majority of 18 patients displayed a consistent protein expression profile and a Ta proteome map was constructed by averaging the grey value of each pixel in all 18 pictures. Spot detection was performed on a project proteome map (based on all 23 samples) and resulted in 1583 detected spots. 416 of these which were positively detected in all 18 "Ta-map" samples. Three patients displayed a pattern with some marked alterations to the majority profile, possibly artefacts of yet unknown heredity. One patient revealed a protein pattern deemed to constitute a separate group, later revealed as a blinded control from a T4 tumour. Only one sample was sparse in protein spots, probably containing mostly blood owing to inadequate sampling. No biological subgroups associated with clinical data were identified. A Ta proteome map was successfully created from TURB samples. Deviating protein expression profiles were identified, indicating a future potential to reveal biologically relevant subgroups in this or other stages of urothelial cell carcinomas.

Identification of NovelListeria monocytogenesSecreted Virulence Factors following Mutational Activation of the Central Virulence Regulator, PrfA

Upon bacterial entry into the cytosol of infected mammalian host cells, the central virulence regulator PrfA of Listeria monocytogenes becomes activated and induces the expression of numerous factors which contribute to bacterial pathogenesis. The mechanism or signal by which PrfA becomes activated during the course of infection has not yet been determined; however, several amino acid substitutions within PrfA (known as PrfA* mutations) that appear to lock the protein into a constitutively activated state have been identified. In this study, the PrfA activation statuses of several L. monocytogenes mutant strains were subjected to direct isogenic comparison and the mutant with the highest activity, the prfA(L140F) mutant, was identified. The prfA(L140F) strain was subsequently used as a tool to identify gene products secreted as a result of PrfA activation. By use of two-dimensional gel electrophoresis followed by liquid chromatography-electrospray ionization-tandem mass spectroscopy analyses, 15 proteins were identified as up-regulated in the prfA(L140F) secretome, while the secretion of two proteins was found to be reduced. Although some of the proteins identified were known to be subject to direct regulation by PrfA, the majority have not previously been associated with PrfA regulation and their expression or secretion may be influenced indirectly by a PrfA-dependent regulatory pathway. Plasmid insertion inactivation of the genes encoding four novel secreted products indicated that three of the four have significant roles in L. monocytogenes virulence. The use of mutationally activated prfA alleles therefore provides a useful approach towards identifying gene products that contribute to L. monocytogenes pathogenesis.

Correlation between Protein Accumulation Profiles and Conventional Toxicological Findings Using a Model Antiandrogenic Compound, Flutamide

In conventional rodent toxicity studies the characterization of the adverse effects of a chemical relies primarily on gravimetric, and histopathological data. The aim of this study was to evaluate if the use of two-dimensional gel electrophoresis could generate protein accumulation profiles, which were in accordance with conventional toxicological findings by investigating a model antiandrogen, flutamide (FM), whose toxic effects, as measured using standard approaches, are well characterized. Male Sprague-Dawley rats were orally exposed to FM (0, 6, 30, and 150 mg/kg/day) for 28 days. The expected inhibition of androgen-dependent tissue stimulation, increased luteinizing hormone and testosterone plasma levels, and Leydig cell hyperplasia were observed. Changes in testicular protein accumulation profiles were evaluated in rats exposed to 150 mg/kg/day FM. Several proteins involved in steroidogenesis (e.g., StAR, ApoE, Hmgcs1, Idi1), cell cycle, and cancer (e.g., Ddx1, Hspd1) were modulated by FM, and these data provided molecular evidence for the hormonal and testicular histopathology changes recorded. Changes in proteins associated with spermatogenesis were also recorded, and these are discussed within the context of the testicular phenotype observed following FM treatment (i.e., normal spermatogenesis but Leydig cell hyperplasia). Overall, our data indicate that the combination of conventional toxicology measurements with omic observations has the potential to improve our global understanding of the toxicity of a compound.

Gel-free and Gel-based Proteomics in Bacillus subtilis: A Comparative Study

The proteome of exponentially growing Bacillus subtilis cells was dissected by the implementation of shotgun proteomics and a semigel-based approach for a particular exploration of membrane proteins. The current number of 745 protein identifications that was gained by the use of two-dimensional gel electrophoresis could be increased by 473 additional proteins. Therefore, almost 50% of the 2500 genes expressed in growing B. subtilis cells have been demonstrated at the protein level. In terms of exploring cellular physiology and adaptation to environmental changes or stress, proteins showing an alteration in expression level are of primary interest. The large number of vegetative proteins identified by gel-based and gel-free approaches is a good starting point for comparative physiological investigations. For this reason a gel-free quantitation with the recently introduced iTRAQ (isobaric tagging for relative and absolute quantitation) reagent technique was performed to investigate the heat shock response in B. subtilis. A comparison with gel-based data showed that both techniques revealed a similar level of up-regulation for proteins belonging to well studied heat hock regulons (SigB, HrcA, and CtsR). However, additional datasets have been obtained by the gel-free approach indicating a strong heat sensitivity of specific enzymes involved in amino acid synthesis.

Quantitative and qualitative differences in protein expression between papillary thyroid carcinoma and normal thyroid tissue

In order to better understand basic mechanisms of tumor development and identify potential new biomarkers, we have performed difference gel electrophoresis (DIGE) and peptide mass fingerprinting on pooled protein extracts from patients with papillary thyroid carcinoma (PTC) compared with matched normal thyroid tissue. Image analysis of DIGE gels comparing PTC and matched normal thyroid tissue protein indicated that 25% of the protein spots were differentially expressed at a 2.5-fold cutoff and 35% at two-fold. Comparison between two different pools of protein from normal thyroid tissues revealed differential protein expression of only 4% at 2.5-fold and 6% at two-fold cutoff. One hundred ninety-two protein spots were identified by MALDI-TOFMS, representing 90 distinct proteins. Excluding albumin, globins and thyroglobulin, imaging software determined 31 proteins to be differentially expressed at the two-fold (or greater) level. Individual gel comparisons (PTC vs. matched normal) from five patients established that 15/31 (48%) of these proteins exhibited statistically significant differential expression. Previously identified molecular markers in this group of proteins include cathepsin B, cytokeratin 19, and galectin-3. Novel differentially expressed proteins include S100A6, moesin, HSP70 (BiP), peroxiredoxin 2, protein phosphatase 2, selenium binding protein 1, vitamin D binding protein, and proteins involved in mitochondrial function. The use of two-dimensional gel electrophoresis (2DGE) revealed a significantly altered protein mass and/or pI in 10%-15% of proteins, suggesting alternatively spliced forms and other posttranslational modification of proteins revealed by this approach. We confirmed S100A6 as a potentially useful biomarker using immunohistochemical analysis (85% sensitivity and 69% specificity for distinguishing benign from malignant thyroid neoplasms). In summary, proteomic analysis of PTC using DIGE and mass spectrometry has confirmed several known biomarkers, uncovered novel potential biomarkers, and provided insights into global pathophysiologic changes in PTC. Many of the differences observed would not have been detected by genomic or other proteomic approaches.

Proteomics of Rac GTPase signaling reveals its predominant role in elicitor-induced defense response of cultured rice cells.

We have previously shown that a human small GTPase Rac homolog, OsRac1, from rice (Oryza sativa) induces cascades of defense responses in rice plants and cultured cells. Sphingolipid elicitors (SEs) have been similarly shown to activate defense responses in rice. Therefore, to systematically analyze proteins whose expression levels are altered by OsRac1 and/or SE treatment, we performed a differential display analysis of proteins by the use of two-dimensional gel electrophoresis and mass spectrometry. A total of 271 proteins whose expression levels were altered by constitutively active (CA)-OsRac1 or SE were identified. Interestingly, of 100 proteins that were up-regulated by a SE, 87 were also induced by CA-OsRac1, suggesting that OsRac1 plays a pivotal role in defense responses induced by SE in cultured rice cells. In addition, CA-OsRac1 induces the expression of 119 proteins. Many proteins, such as pathogenesis-related proteins, SGT1, and prohibitin, which are known to be involved in the defense response, were found among these proteins. Proteins involved in redox regulation, chaperones such as heat shock proteins, BiP, and chaperonin 60, proteases and protease inhibitors, cytoskeletal proteins, subunits of proteasomes, and enzymes involved in the phenylpropanoid and ethylene biosynthesis pathways were found to be induced by CA-OsRac1 or SE. Results of our proteomic analysis revealed that OsRac1 is able to induce many proteins in various signaling and metabolic pathways and plays a predominant role in the defense response in cultured rice cells.

Proteome analysis of a recombinant Bacillus megaterium strain during heterologous production of a glucosyltransferase

A recombinant B. megaterium strain was used for the heterologous production of a glucosyltransferase (dextransucrase). To better understand the physiological and metabolic responses of the host cell to cultivation and induction conditions, proteomic analysis was carried out by combined use of two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) for protein separation and identification.2-DE method was optimized for the separation of intracellular proteins. Since the genome of B. megaterium is not yet available, peptide sequencing using peptide fragment information obtained from nanoelectrospray ionization quadrupole-time-of-flight tandem mass spectrometry (ESI-QqTOF MS/MS) was applied for protein identification. 167 protein spots were identified as 149 individual proteins, including most enzymes involved in the central carbon metabolic pathways and many enzymes related to amino acid synthesis and protein synthesis. Based on the results a 2-DE reference map and a corresponding protein database were constructed for further proteomic approaches on B. megaterium.For the first time it became possible to perform comparative proteomic analysis on B. megaterium in a batch culture grown on glucose with xylose induction for dextrasucrase production. No significant differences were observed in the expression changes of enzymes of the glycolysis and TCA cycle, indicating that dextransucrase production, which amounted to only 2 % of the entire protein production, did not impose notable metabolic or energetic burdens on the central carbon metabolic pathway of the cells. However, a short-term up-regulation of aspartate aminotransferase, an enzyme closely related to dextransucrase production, in the induced culture demonstrated the feasibility to use 2-DE method for monitoring dextransucrase production. It was also observed that under the cultivation conditions used in this study B. megaterium tended to channel acetyl-CoA into pathways of polyhydroxybutyrate production. No expression increases were found with cytosolic chaperones such as GroEL and DnaK during dextransucrase production and secretion, whereas a strong up-regulation of the oligopeptide-binding protein OppA was observed in correlation with an increased secretion of dextransucrase into the culture medium.

Metabolomic and proteomic biomarkers for III–V semiconductors: Chemical-specific porphyrinurias and proteinurias

A pressing need exists to develop and validate molecular biomarkers to assess the early effects of chemical agents, both individually and in mixtures. This is particularly true for new and chemically intensive industries such as the semiconductor industry. Previous studies from this laboratory and others have demonstrated element-specific alterations of the heme biosynthetic pathway for the III–V semiconductors gallium arsenide (GaAs) and indium arsenide (InAs) with attendant increased urinary excretion of specific heme precursors. These data represent an example of a metabolomic biomarker to assess chemical effects early, before clinical disease develops. Previous studies have demonstrated that the intratracheal or subcutaneous administration of GaAs and InAs particles to hamsters produces the induction of the major stress protein gene families in renal proximal tubule cells. This was monitored by 35-S methionine labeling of gene products followed by two-dimensional gel electrophoresis after exposure to InAs particles. The present studies examined whether these effects were associated with the development of compound-specific proteinuria after 10 or 30 days following subcutaneous injection of GaAs or InAs particles in hamsters. The results of these studies demonstrated the development of GaAs- and InAs-specific alterations in renal tubule cell protein expression patterns that varied at 10 and 30 days. At the 30-day point, cells in hamsters that received InAs particles showed marked attenuation of protein expression, suggesting inhibition of the stress protein response. These changes were associated with GaAs and InAs proteinuria patterns as monitored by two-dimensional gel electrophoresis and silver staining. The intensity of the protein excretion patterns increased between the 10- and 30-day points and was most pronounced for animals in the 30-day InAs treatment group. No overt morphologic signs of cell death were seen in renal tubule cells of these animals. Western blot analyses of the urines with antibodies to the 32-, 70-, and 90-kDa stress protein families did not show the presence of these molecules, indicating that these proteins were not excreted in the urine samples. These data suggest that the observed proteinuria patterns were not a result of cell death and that the observed chemical-specific proteinurias were produced before marked cellular toxicity. These findings suggest a hypothesis involving GaAs and InAs interference with stress protein chaperoning of reabsorbed proteins for proteosomic degradation and the probable chaperoning of damaged intracellular proteins from renal proximal tubule cells into the urinary filtrate. Overall, the results of these studies provide further information on the nephrotoxicity of these semiconductor compounds. They also suggest the use of two-dimensional gel electrophoresis with silver staining of urinary protein patterns as a potentially useful proteomic approach to renal damage early in relation to intracellular proteotoxicity in kidney tubule cells.

Use of two-dimensional gel electrophoresis in predictive toxicology: Identification of potential early protein biomarkers in chemically induced hepatocarcinogenesis

Our current approach focused on the identification of potential early protein biomarker signatures which are indicative of the carcinogenic processes in rats exposed to 20 mg/kg of the liver carcinogen N-nitrosomorpholine (NNM). Treated liver was investigated at different timepoints. Therefore, proteins were separated by two-dimensional gel electrophoresis as a first step prior to identification of differentially expressed proteins by mass spectrometry. Proteomic analysis of liver samples after one day of exposure revealed significant upregulation of proteins involved in response to cellular stress induced by NNM (superoxide dismutase, heat shock protein 60, peroxiredoxin). Eighteen weeks after withdrawal of NNM, we were able to identify cancer-related proteins in rat liver bearing malignant, transformed cells (caspase-8 precursor, vimentin, Rho GDP dissociation inhibitor). Some of these proteins were already deregulated after three weeks of exposure indicating their potential usefulness as early predictive biomarkers for liver carcinogenicity (annexin A5, fructose-1,6-bisphosphatase). As regulatory toxicology approaches usually include the investigation of carcinogenicity in two-years studies in rodents, especially the detection of early protein biomarker signatures which precede the appearance of neoplasia, demonstrates the high potential of proteomics approaches to substantially reduce the time and costs of carcinogenicity testing.

Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/ΔF508‐CFTR to the plasma membrane

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF.

Improvement of the two‐dimensional gel electrophoresis analysis for the proteome study of Halobacterium salinarum

Inherent problems exist in the use of two-dimensional gel electrophoresis (2-DE) for sample preparation and separation of proteins from Halobacterium salinarum. In particular, proteins from cells grown in 25% NaCl are difficult to resolve by 2-DE due to the abundance of salt. To remove salts, a 3 kDa molecular weight cut-off column was used. When soluble proteins were separated by 2-DE, most of the proteins were concentrated in the acidic range. For separation of proteins in the pH 3-6 range, ultrazoom immobilized pH gradient strips were used. In addition, sample separation using a IPGphor/Multiphor combined system was a more effective method for the proteome analysis of acidic proteins than using IPGphor for the isoelectric focusing step.

The characterisation of novel secreted Ly-6 proteins from rat urine by the combined use of two-dimensional gel electrophoresis, microbore high performance liquid chromatography and expressed sequence tag data.

A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.