Acute assessment of an aspalathin-enriched green rooibos (Aspalathus linearis) extract with hypoglycemic potential.

Rooibos, an endemic South African plant, known for its use as herbal tea, has potential as an antidiabetic herbal product, following recent demonstration of the glucose lowering effect of its major flavonoid, the dihydrochalcone C-glucoside aspalathin. The purpose of this study was to confirm antidiabetic activity for rooibos extract high in aspalathin content. An extract (SB1) was selected after screening for high aspalathin content and α-glucosidase inhibition activity. On-line HPLC-biochemical detection confirmed α-glucosidase inhibitory activity for aspalathin. In vitro the extract induced a dose response increase in glucose uptake (5 × 10⁻5 to 5 µg/ml) on C2C12 myotubules. Aspalathin was effective at 1, 10 and 100 µM, while rutin was effective at 100 µM. In the Chang cells only the extract was effective. In vivo the extract sustained a glucose lowering effect comparable to metformin over a 6h period after administration (25mg/kg body weight (BW)) to STZ-induced diabetic rats. In an oral glucose tolerance test the extract (30 mg/kg BW) was more effective than vildagliptin (10mg/kg BW), a dipeptidyl peptidase-4 inhibitor. An aspalathin-rutin mixture (1:1; m/m) dosed at 1.4 mg/kg BW, but not the single compounds separately, reduced blood glucose concentrations of STZ-induced diabetic rats over a 6h monitoring period. The improved hypoglycemic activity of the aspalathin-rutin mixture and the extract illustrated synergistic interactions of polyphenols in complex mixtures.

Changes in expression of IL-1 beta influenced proteins in transplanted islets during development of diabetes in diabetes-prone BB rats.

AIMS/HYPOTHESIS: Type 1 diabetes mellitus is a multifactorial autoimmune disease characterised by selective destruction of beta cells in the islets of Langerhans. We have previously shown that IL-1 beta modulates beta cell function, causes beta cell death and induces expression changes in 82 out of 1815 protein spots detected by two-dimensional gel electrophoresis (2-DGE) in diabetes-prone bio-breeding (BB-DP) rat islets in vitro. The aim of this study was to describe the relevance of these proteins in the development of diabetes in vivo. METHODS: Syngeneic neonatal islets ( n=200) were transplanted under the kidney capsule of 30-day-old BB-DP and control rats, removed to different time points after transplantation or at the onset of diabetes, and metabolically labelled with S(35)-methionine for 2-DGE. The 82 proteins were re-localised and followed. In addition, transplants were examined for expression of IL-1 beta mRNA by in situ hybridisation. RESULTS: All 82 proteins could be re-localised in all syngeneic transplants from BB-DP and control rats. A total of 60 of the 82 proteins were changed during development of diabetes. Of the 82 proteins, 32 were changed in expression at the onset of diabetes compared to non-diabetic BB-DP rats, and 25 of these were changed as by IL-1 beta in vitro. Highest expression of IL-1 beta mRNA was found at the onset of diabetes. CONCLUSIONS/INTERPRETATION: IL-1 beta-induced protein expression changes in islets in vitro also occur in vivo and change in a complex pattern during the development of diabetes in the BB-DP rat. No single protein seems to be responsible for the development of diabetes, but rather the cumulative numbers of changes seem to interfere with the intracellular stability of the beta cell.

Protein expression changes in a cell system of beta-cell maturation reflect an acquired sensitivity to IL-1beta.

AIM/HYPOTHESIS: Type 1 diabetes mellitus (T1DM) is caused by specific destruction of the pancreatic beta cells in the islets of Langerhans. Increased sensitivity to cytokines, in particular to interleukin-1beta (IL-1beta) seems to be an acquired trait during beta-cell maturation. In response to cytokines both protective and deleterious mechanisms are induced in beta cells, and when the deleterious prevail, T1DM develops. The aims of this study were to identify perturbation in protein patterns (PiPP) associated with beta-cell maturation, and compare these changes to previous analyses of IL-1beta exposed rat islets. For this purpose, proteome analyses were carried out using a cell-line, which matures from a glucagon-producing pre-beta-cell phenotype (NHI-glu) to an insulin-producing beta-cell phenotype (NHI-ins). We have previously shown that this maturation is accompanied by acquired sensitivity to the toxic effects of IL-1beta. METHODS: 2D-gel electrophoresis was used to separate the proteins and MALDI-MS and database searches were performed to identify the proteins. RESULTS: During beta-cell maturation 135 protein spots out of 2239 detectable changed expression levels. Of these, 74 were down-regulated, 44 up-regulated, 16 were suppressed and 1 was expressed de novo. Using MALDI-MS, positive identification was obtained for 93 out of the 135 protein-spots revealing 97 different proteins. Of these, 22 proteins were in common with changes identified in previous proteome analysis of perturbation in protein pattern in IL-1beta exposed rat islets. Several of the proteins were present in more than one spot suggesting post-translational modification. CONCLUSION/INTERPRETATION: Several proteins and protein modifications were identified that could be critically involved in beta-cell maturation, insulin-gene expression and the acquired IL-1beta sensitivity.

Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans.

Streptococcus mutans is a component of the dental plaque biofilm and a major causal agent of dental caries. Log-phase cells of the organism are known to induce an acid tolerance response (ATR) at sub-lethal pH values ( approximately 5.5) that enhances survival at lower pH values such as those encountered in caries lesions. In this study, we have employed a rod biofilm chemostat system to demonstrate that, while planktonic cells induced a strong ATR at pH 5.5, biofilm cells were inherently more acid resistant than such cells in spite of a negligible induction of an ATR. Since these results suggested that surface growth itself triggered an ATR in biofilm cells, we were interested in comparing the effects of a pH change from 7.5 to 5.5 on protein synthesis by the two cell types. For this, cells were pulse labeled with [(14)C]-amino acids following the pH change to pH 5.5, the proteins extracted and separated by two-dimensional (2D) electrophoresis followed by autoradiography and computer-assisted image analysis. A comparison between the cells incubated at pH 5.5 and the control biofilm cells revealed 23 novel proteins that were absent in the control cells, and 126 proteins with an altered relative rate of synthesis. While the number of changes in protein expression in the biofilm cells was within the same range as for planktonic cells, the magnitude of their change was significantly less in biofilm cells, supporting the observation that acidification of biofilm cells induced a negligible ATR. Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregulation of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment. On the other hand, the glycolytic enzymes in control biofilm cells were significantly less downregulated and key enzymes, such as lactate dehydrogenase were upregulated during pH 5.5 incubation, suggesting that the enhanced acid resistance of biofilm cells is associated with the maintenance of pH homeostasis by H+ extrusion via membrane ATPase and increased lactate efflux.

Intrauterine programming of fetal islet gene expression in rats--effects of maternal protein restriction during gestation revealed by proteome analysis.

AIMS/HYPOTHESIS: Fetal undernutrition can result in intrauterine growth restriction and increased incidence of Type 2 diabetes mellitus. Intrauterine malnutrition in form of an isocaloric low-protein diet given to female rats throughout gestation decreases islet-cell proliferation, islet size and pancreatic insulin content, while increasing the apoptotic rate and sensitivity to nitrogen oxide and interleukin-1beta. Hence, the influence of a low-protein diet on the development of beta-cells and islets could also be of interest for the pathogenesis of Type 1 and Type 2 diabetes mellitus. We hypothesise that the effects of a low-protein diet in utero are caused by intrauterine programming of beta-cell gene expression. METHODS: Pregnant Wistar rats were fed a low-protein diet (8% protein) or a control diet (20% protein) throughout gestation. At day 21.5 of gestation fetal pancreata were removed, digested and cultured for 7 days. Neoformed islets were collected and analysed by proteome analysis comprising 2-dimensional gel electrophoresis and mass spectrometry. RESULTS: A total of 2810 different protein spots were identified, 70 of which were changed due to the low-protein diet. From 45 of the changed protein spots, identification was obtained by mass spectrometry (64% success rate). Proteins induced by the low-protein diet were grouped according to their biological functions, e.g. cell cycle and differentiation, protein synthesis and chaperoning. CONCLUSIONS/INTERPRETATION: Our study offers a possible explanation of the alterations induced by a low-protein diet in islets. It shows that in Wistar rats the intrauterine milieu could program islet gene expression in ways unfavourable for the future of the progeny. This could be important for our understanding of the development of Type 1 and Type 2 diabetes mellitus.

IL-1beta induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis.

AIMS/HYPOTHESIS: Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1beta (IL-1beta) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1beta exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen in IL-1beta exposed Wistar Furth (WF) rat islets. METHODS: The 82 protein spots, that changed expression after IL-1beta exposure, were all re-identified on preparative gels of 200 000 neonatal WF rat islets, cut out and subjected to mass spectrometry for identification. RESULTS: Forty-five different proteins were identified from 51 spots and grouped according to function: (i) energy transduction and redox potentials; (ii) glycolytic and Krebs cycle enzymes; (iii) protein, DNA and RNA synthesis, chaperoning and protein folding; (iv) signal transduction, regulation, differentiation and apoptosis; (v) cellular defence; and (vi) other functions. Comparison of IL-1beta exposed BB-DP and WF islets showed common changes in 14 proteins and several proteins influencing similar pathways, suggesting that similar routes in the two strains lead to beta-cell destruction. CONCLUSION/INTERPRETATION: We demonstrate that proteome analysis is a powerful tool to identify proteins and pathways in BB-DP rat islets exposed to IL-1beta.

Phospho-proteomics: evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis.

Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in such proteins is a major challenge in proteomics. In the present study we evaluate the use of enzymatic de-phosphorylation in combination with differential peptide mass mapping for identification of phosphorylated peptides in peptide mixtures derived from in-gel digested phospho-proteins. Phospho-peptides could be identified provided that improved sample preparation methods prior to mass spectrometric analysis were used. An attempt to identify the proteins visualized by [32P] autoradiography in a proteomics study and their phosphorylation sites, demonstrated that protein identification was possible whereas reliable identification of the phospho-peptides requires more protein than normally available in our proteomics studies.

The effects of transcription regulating genes PDR1, pdr1-3 and PDR3 in pleiotropic drug resistance.

Mutations in the yeast PDR1 or PDR3 genes lead to acquisition of resistance towards various unrelated cytotoxic compounds. The broad range and different properties of these compounds indicate the existence of mechanisms which protect cellular targets, neutralise or expel the compounds from the cell. In wild type and pdr mutants, 83 proteins, out of 2706 detected by two-dimensional gel electrophoresis, were differentially expressed. Fifty-three of these could be identified by mass spectrometry. The functions of these 53 proteins fall into several metabolic groups demonstrating that drug resistance phenotype is a mosaic response derived from such diverse functions as stress defence, endocytosis, oxidation and reduction, amino acid synthesis and mitochondrial biogenesis. The patterns of synthesis of the selected proteins clearly demonstrates the complex interaction between Pdr1p and Pdr3p in exerting their regulatory functions. The data also indicate that, in the Saccharomyces cerevisiae pleiotropic drug resistance phenomenon, translational events exert a more decisive effect than transcription in regulating the levels of active forms of the proteins involved.

Proteome analysis of interleukin-1beta--induced changes in protein expression in rat islets of Langerhans.

The intracellular molecular events involved in the beta-cell death process are complex but poorly understood. Cytokines, e.g., interleukin (IL)-1beta, may play a crucial role in inducing this process. Protein synthesis is necessary for the deleterious effect of IL-1, and induction of both protective and deleterious proteins has been described. To characterize the rather complex pattern of islet protein expression in rat islets in response to IL-1, we have attempted to identify proteins of altered expression level after IL-1 exposure by 2D gel electrophoresis and mass spectrometry. Of 105 significantly changed (i.e., up- or downregulated or de novo-induced) protein spots, we obtained positive protein identification for 60 protein spots. The 60 identifications corresponded to 57 different proteins. Of these, 10 proteins were present in two to four spots, suggesting that posttranslatory modifications had occurred. In addition, 11 spots contained more than one protein. The proteins could be classified according to their function into the following groups: 1) energy transduction; 2) glycolytic pathway; 3) protein synthesis, chaperones, and protein folding; and 4) signal transduction, regulation, differentiation, and apoptosis. In conclusion, valuable information about the molecular mechanisms involved in cytokine-mediated beta-cell destruction was obtained by this approach.

Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry.

Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis. The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis. In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated. A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2. The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1). Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.

2D or not 2D. Two-dimensional gel electrophoresis.

2D gel electrophoresis is the technology that everyone loves to hate-it requires manual dexterity and precision to reproduce precisely and is thus not well-suited as a high-throughput technology. Although almost everyone would like to replace it, the resolution and sensitivity it offers are exquisite and unsurpassed if one wants a global view of cellular activity. There have been several recent developments, for example, the detection of low abundance proteins, and the resolution possible with narrow-range IPG gels.

Comparison of the proteomes of three yeast wild type strains: CEN.PK2, FY1679 and W303.

Yeast deletion strains created during gene function analysis projects very often show drastic phenotypic differences depending on the genetic background used. These results indicate the existence of important molecular differences between the CEN.PK2, FY1679 and W303 wild type strains. To characterise these differences we have compared the protein expression levels between CEN.PK2, FY1679 and W303 strains using twodimensional gel electrophoresis and identified selected proteins by mass spectrometric analysis. We have found that FY1679 and W303 strains are more similar to each other than to the CEN.PK2 strain. This study identifies 62 proteins that are differentially expressed between the strains and provides a valuable source of data for the interpretation of yeast mutant phenotypes observed in CEN.PK2, FY1679 and W303 strains.

Cytokine- or chemically derived nitric oxide alters the expression of proteins detected by two-dimensional gel electrophoresis in neonatal rat islets of Langerhans.

Interleukin-1beta (IL-1beta) treatment of neonatal rat islets for 24 h induces changes in the expression of 105 of 2,200 proteins, as determined previously by two-dimensional (2D) gel electrophoresis. Nitric oxide (NO) has been implicated as one of the mediators of IL-1beta effects in insulin-containing cell lines and rat islets. The aims of this study were 1) to determine the involvement of NO in IL-1beta-induced alterations in protein expression and 2) to investigate the effects of chemically generated NO on protein expression by 2D gel electrophoresis of neonatal rat islet samples. IL-1beta-induced NO production was prevented by incubation of islets in arginine-free medium supplemented with the arginine analog NG-nitro-L-arginine. [35S]methionine-labeled islet proteins were separated using 2D gel electrophoresis and analyzed using the BioImage computer program. Analysis revealed that the expression levels of 23 protein spots of the 105 protein spots, altered by prior treatment with IL-1beta (60 U/ml) alone, were significantly affected (P < 0.01 [n = 4] and P < 0.05 [n = 19]) when NO production was prevented. The effects of chemically generated NO were investigated by exposing islets to the NO donor GSNO (100 micromol/l) for 24 h before labeling with [35S]methionine and 2D gel electrophoresis. Computer-based analysis identified alterations in the expression of 19 of a total of 1,600 detectable proteins in GSNO-treated islets (P < 0.01). We conclude 1) that the expression of up to 42 proteins is altered by cytokine-induced or chemically generated NO in the precise experimental conditions chosen and 2) that the majority of proteins altered by prior treatment with IL-1beta may be the result of NO-independent IL-1beta-mediated regulation of gene expression. This study demonstrates that the combination of 2D gel electrophoresis and mass spectrometry is a powerful tool in the identification of beta-cell proteins involved in the response to toxic mediators.

Islet protein expression changes during diabetes development in islet syngrafts in BB-DP rats and during rejection of BB-DP islet allografts.

Interleukin 1beta (IL-1) is cytotoxic to rat pancreatic beta-cells in vitro, and increased expression of IL-1 mRNA is found in the islets of Langerhans during development of diabetes in BB/Wor/Mol-BB2 (BB-DP) rats and NOD mice. It has been proposed that IL-1 induces a race between protective and deleterious proteins in the beta-cells during development of diabetes, and that heat shock proteins 70 and 90, and manganese superoxide dismutase, all inducible by IL-1 are potentially protective proteins. We have established a database of approximately 2000 neonatal rat-islet proteins by two-dimensional gel (2-D gel) electrophoresis of [35S]-methionine labelled neonatal Wistar Furth rat islets. In these IL-1 was shown to up- or down-regulate the islet-expression level of 99, and to induce de novo synthesis of 6 proteins. The identity of most of the IL-1 induced proteins is unknown and under study. In this study we wished to investigate if changes in protein expression induced in vitro by IL-1 stimulation of islets are also seen in vivo during spontaneous development of diabetes in BB-DP rats, and during islet allograft rejection. Two-hundred neonatal BB-DP rat islets were grafted under the kidney capsule of either 30-day-old BB-DP rats killed at onset of diabetes or of 30-day-old Wistar Kyoto (WK) rats, killed 12 days after grafting. Proteins in excised islet-grafts and in vitro IL-1 exposed isolated neonatal BB-DP rat islets were labelled with [35S]-methionine, and processed for 2-D gel electrophoresis. Fluorographs of the gels were analysed by computer. A total of 1815 proteins were found in 3 of 3 12.5% polyacrylamide gels. Interleukin-1 was found to change expression level of 82 of these proteins (22 up- and 60 down-regulated) in neonatal BB-DP rat islets in vitro. Of these 82 proteins 33 (4 up- and 29 down-regulated) also changed level of expression during disease occurrence in syngeneic islet grafts from diabetic BB-DP rats, and 29 (4 up- and 25 down-regulated) during rejection of BB-DP islets grafted to WK rats. Changes in the expression level of 14 (3 up- and 11 down-regulated) of the 82 proteins altered by IL-1 in vitro were only found in syngeneic islet grafts in diabetic BB-DP rats, and changes in the expression level of 8 (2 up- and 6 down-regulated) of these 82 proteins expression were only found in BB-DP islet allografts in WK recipients. Identification of these proteins may be important in understanding the mechanisms of islet destruction during development of insulin-dependent diabetes mellitus and during islet allograft rejection.

Towards higher resolution: two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients.

The rising number of proteome projects leads to new challenges for two-dimensional electrophoresis with immobilized pH gradients and different applications of this technique. Not only wide pH gradients such as 4-12 or 3-12 (Görg et al., Electrophoresis 1999, 20, 712-717) which can give an overview of the total protein expressions of cells are in demand but also overlapping narrow immobilized pH gradients are to be used for more specialized and detailed research and micropreparative separations. The advantage of overlapping narrow pH gradients is the gain in higher resolution by stretching the protein pattern in the first dimension. This simplifies computer-aided image analysis and protein identification (e.g., by mass spectrometry). In this study the protein patterns of yeast cells in pH gradients 4-5, 4.5-5.5, 5-6, 5.5-6.7 and 6-9 are presented and compared to the pH 4-7 and 3-10 gradients. This combination allowed us to reveal a total of 2286 yeast protein spots compared to 755 protein spots in the pH 3-10 gradient.

Two-dimensional gel analysis of human endometrial proteins: characterization of proteins with increased expression in hyperplasia and adenocarcinoma.

In the search for new markers of human endometrial hyperplasia and adenocarcinoma the method of quantitative two-dimensional gel electrophoresis was applied to study the protein expression profiles of metabolically [(35)S]-methionine-labelled proteins of endometrial explants. Approximately 1700 protein spots were resolved by the two-dimensional gel electrophoresis, and the expression pattern of each of these proteins was assessed for increased expression during hyperplasia or adenocarcinoma. In total, six protein spots showed increased expression in hyperplasia, 19 in carcinoma, and eight in both hyperplasia and carcinoma. Twelve of these 33 differentially expressed proteins were identified by peptide mass mapping combined with sequence database searching. Among the identified proteins were proteins involved in cellular transport and chaperoning, i.e. heat shock protein 27 kDa protein, heat shock 70 kDa protein, heat shock cognate 71 kDa protein, and serotransferrin. Other identified proteins were: regulatory chain protein of cAMP-dependent protein kinase, prohibitin, and heterogeneous nuclear ribonucleoprotein A2/B1. Finally we identified proteins associated with the cytoskeleton, vimentin and tropomyosin isoform 3, and the glycolytic pathway, alpha enolase, and phosphoglycerate kinase. The remaining unidentified proteins were either not contained in the database and must be assumed to be novel proteins, or were present in too low amounts to allow characterization.

Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis.

Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea/ 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high Mr proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii).

Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification.

Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross-references demonstrated that there is no obvious pattern by which the mobility of a protein in one gel system can be used to predict its mobility in the other. Thus, as laboratories adopt the immobilized pH gradient-based 2-D gel systems, the only reliable means of translating the data gained with the carrier ampholyte-based gel system is to positively identify the proteins in both 2-D systems.

Proteome analysis of Saccharomyces cerevisiae: a methodological outline.

Proteome analysis offers a unique means of identifying important proteins, characterizing their modifications and beginning to describe their function. This is achieved through the combination of two technologies: protein separation and selection by two-dimensional gel electrophoresis, and protein identification and characterization by mass spectrometry. This methodological outline sketches the strengths and weaknesses of the two central technologies used, and provides both practical tips and the theoretical background for their utilization. One application of these technologies is illustrated by the characterization of genes, revealed by sequencing, but which have no--or only weak homology--to any other known genes. Other applications, for example the identification of protein markers for particular human diseases, are only referred to. The aim of the article is thus to provide the basis for a sound understanding of the full potential and limitations of proteome analysis.

Interleukin-1beta induced changes in the protein expression of rat islets: a computerized database.

Insulin-dependent diabetes mellitus is caused by an autoimmune destruction of the beta-cells in the islets of Langerhans. The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to beta-cells in isolated pancreatic rat islets. The antigen(s) triggering the immune response as well as the intracellular mechanisms of action of interleukin 1-mediated beta-cell cytotoxicity are unknown. However, previous studies have found an association of beta-cell destruction with alterations in protein synthesis. Thus, two-dimensional (2-D) gel electrophoresis of pancreatic islet proteins may be an important tool facilitating studies of the molecular pathogenesis of insulin-dependent diabetes mellitus. 2-D gel electrophoresis of islet proteins may lead to (i) the determination of qualitative and quantitative changes in specific islet proteins induced by cytokines, (ii) the determination of the effects of agents modulating cytokine action, and (iii) the identification of primary islet protein antigen(s) initiating the immune destruction of the beta-cells. Therefore, the aim of this study was to create databases (DB) of all reproducibly detectable protein spots on 10% and 15% acrylamide 2-D gels of neonatal rat islets (10% and 15% DB), labeled under standardized culture conditions. 1235 and 557 spots were present in 5 of 5 gels in the 15% isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) DB, respectively, whereas 995 and 378 spots were present in 5 of 5 gels in the 10% IEF and NEPHGE DB, respectively, yielding a reproducibility of spot detection between 75.2% and 91.7%. In both DBs, the average coefficient of variation of the percentage of integrated optical density (CV% of %IOD) for spots present in all gels was between 42.4% and 45.7%. When the same sample was analyzed in consecutive sets of gels on different days (interassay analysis), the average CV% of %IOD was 35.5%-36.1%. When the same sample was analyzed repeatedly in one set of gels (intra-assay analysis), the average CV% of %IOD was 30.2% in the IEF gels, while the average CV% of %IOD was 45.7% in the NEPHGE gels. Addition of interleukin-1beta (IL-1beta) to the cultures resulted in statistically significant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2-D gel protein databases of neonatal rat islets of Langerhans and demonstrate its usage to identify proteins altered in expression by IL-1beta.