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Although proteomic technologies have been enthusiastically embraced in the field of biomarker discovery and particularly with biological fluids, it is increasingly being recognized that pre-analytical effects, i.e. those occurring prior to the point of actual sample analysis and including factors such as sample processing and storage, can exert marked influences on the results obtained. Such effects have been recognized already for specific analytes in clinical chemistry, but with the increasing sensitivity and resolution of the newer technologies, such effects are potentially even more marked. The challenge of translating initial findings into clinical application requires such issues to be addressed at a very early stage in study design and this paper reviews the current knowledge of the potential impact of pre-analytical factors on proteomic studies of biological fluids.

With a mean weight of 1500 g containing around 10 billion neurons, the adult brain represents about 2% of the total body mass, but requires 20% of the total energy produced. It consumes continuously 150 g of glucose and 72 L of oxygen every 24 h. A few minutes interruption of this supply can lead to dramatic brain damage. Manifestations and consequences of stroke depend on the location and extent of the lesions. A vascular cerebral accident, also called stroke or brain attack, is an interruption of the blood supply owing to either occlusion (ischemic stroke) or rupture (hemorrhagic stroke) of a blood vessel to any part of the brain, with an occurrence of around 80% for the ischemic type. Stroke has a devastating impact on public health and remains the third leading cause of death and the first leading cause of long-term disability in industrialized countries. An early diagnosis of the cerebral accident associated with an appropriate treatment would reduce the risk of death and enhance the chances of recovery. When the diagnosis of stroke is established, the physician needs to know the nature (ischemic or hemorrhagic), the extent, and the location of the accident in order to orient patients and to give them most suitable treatment. Because no specific and unique symptoms or early blood diagnostic markers are currently available, it was of a great interest to develop new approaches in the research and discovery area of new early diagnosis and prognosis markers of stroke.

Background To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. Results In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over-expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper-mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. Conclusion Our proteome and transcriptome analyses have been performed during stationary-phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets.

The aim of this manuscript is to initiate a constructive discussion about the definition of clinical proteomics, study requirements, pitfalls and (potential) use. Furthermore, we hope to stimulate proposals for the optimal use of future opportunities and seek unification of the approaches in clinical proteomic studies. We have outlined our collective views about the basic principles that should be considered in clinical proteomic studies, including sample selection, choice of technology and appropriate quality control, and the need for collaborative interdisciplinary efforts involving clinicians and scientists. Furthermore, we propose guidelines for the critical aspects that should be included in published reports. Our hope is that, as a result of stimulating discussion, a consensus will be reached amongst the scientific community leading to guidelines for the studies, similar to those already published for mass spectrometric sequencing data. We contend that clinical proteomics is not just a collection of studies dealing with analysis of clinical samples. Rather, the essence of clinical proteomics should be to address clinically relevant questions and to improve the state-of-the-art, both in diagnosis and in therapy of diseases.

2-DE remains the most popular and versatile protein separation method among a rapidly growing array of various proteomics technologies. However, variability in sample processing, experimental design and data analyses results in a limited cross-validation between studies performed in different laboratories. One of the goals of the Human Proteome Organization (HUPO) is to establish standards and guidelines for proteomics studies. We contributed to the HUPO Brain Proteome Project by analyzing brains from neonatal and adult mice using 2-DE. Here we propose a standard workflow to analyze 2-DE images and extract statistically significant differences. After differential analysis and identification by MALDI-TOF/TOF, dihydropyrimidinase-related proteins, brain FABP, stathmin, isocitrate dehydrogenase, gamma enolase, annexin V, glutamine synthetase, creatine kinase B chain, triosephosphate dehydrogenase, and malate dehydrogenase were found differentially expressed between the two groups. The functions and potential mechanisms underlying the variation observed for these proteins are discussed.

The proteome of the human nucleolus was investigated in a single analysis using off-line strong cation exchange chromatography and microfraction collection combined with HPLC-chip/MS. The analysis was conducted either as a 1-D workflow with HPLC-chip alone or as a 2-D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2-D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1-D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false-positive identifications, were applied for reliable protein identification.

OBJECTIVE: To determine whether pharmacogenetic tests such as N-acetyltransferase 2 (NAT2) and cytochrome P450 2E1 (CYP2E1) genotyping are useful in identifying patients prone to antituberculosis drug-induced hepatotoxicity in a cosmopolite population. METHODS: In a prospective study we genotyped 89 patients treated with isoniazid (INH) for latent tuberculosis. INH-induced hepatitis (INH-H) or elevated liver enzymes including hepatitis (INH-ELE) was diagnosed based on the clinical diagnostic scale (CDS) designed for routine clinical practice. NAT2 genotypes were assessed by fluorescence resonance energy transfer probe after PCR analysis, and CYP2E1 genotypes were determined by PCR with restriction fragment length polymorphism analysis. RESULTS: Twenty-six patients (29%) had INH-ELE, while eight (9%) presented with INH-H leading to INH treatment interruption. We report no significant influence of NAT2 polymorphism, but we did find a significant association between the CYP2E1 *1A/*1A genotype and INH-ELE (OR: 3.4; 95% CI:1.1-12; p = 0.02) and a non significant trend for INH-H (OR: 5.9; 95% CI: 0.69-270; p = 0.13) compared with other CYP2E1 genotypes. This test for predicting INH-ELE had a positive predictive value (PPV) of 39% (95% CI: 26-54%) and a negative predictive value (NPV) of 84% (95% CI: 69-94%). CONCLUSION: The genotyping of CYP2E1 polymorphisms may be a useful predictive tool in the common setting of a highly heterogeneous population for predicting isoniazid-induced hepatic toxicity. Larger prospective randomized trials are needed to confirm these results.

Prostaglandin D2 synthase (PGDS) (beta-trace protein) is a highly abundant cerebrospinal fluid (CSF) glycoprotein. A number of studies have been performed to determine the potential value of this protein for the diagnosis of various neurological disorders. The measurement of total PGDS levels in CSF has proved marginally useful for this purpose, but promising results were obtained while investigating changes in the posttranslational modifications (PTM) pattern. Using 2-DE analysis, we previously showed that PGDS is differentially expressed in ante- and post mortem CSF samples. In the present study, we examined whether the PGDS isoforms may help to distinguish stroke and neurodegenerative disease patients from healthy subjects. The pattern of PGDS PTM was analyzed in CSF from patients with various neurological disorders (n = 44) using IEF/immunoblotting techniques. Strong alterations of this pattern were detected in patients with different forms of degenerative dementia. These findings are consistent with PGDS being altered in some neurological diseases and provide new opportunities for clinical applications.

Heart-fatty acid-binding protein (H-FABP) is a small cytosolic protein involved in intracellular fatty acid transport. This protein, highly concentrated in the heart, is quickly released into plasma after myocardial injury. Results from numerous studies suggest that H-FABP is an excellent marker for the early detection of myocardial damage. H-FABP is also expressed in the brain, although in lower concentrations than in the heart. Recent preliminary studies also investigated the usefulness of H-FABP for the diagnosis of acute and chronic neurological disorders. These potential applications of H-FABP in clinical practice are reviewed in this article, with a strong focus on the early diagnosis of acute myocardial infarction and stroke.

Acquisition of tandem mass spectra from peptides or other analytes deposited on non-conductive membranes is inhibited on instruments combining matrix-assisted laser desorption/ionization with tandem time-of-flight analyzers (MALDI-TOF/TOF) due to a charging effect. A thin layer of gold renders the membrane conductive. This allows adequate data acquisition on MALDI-TOF/TOF systems. Therefore, this methodology extends the capacity of the molecular scanner concept to tandem mass spectrometry.

Images obtained from high-throughput mass spectrometry (MS) contain information that remains hidden when looking at a single spectrum at a time. Image processing of liquid chromatography-MS datasets can be extremely useful for quality control, experimental monitoring and knowledge extraction. The importance of imaging in differential analysis of proteomic experiments has already been established through two-dimensional gels and can now be foreseen with MS images. We present MSight, a new software designed to construct and manipulate MS images, as well as to facilitate their analysis and comparison.

The phosphorylation of heat shock protein 27 (HSP27) occurs differently in human renal cell carcinoma (RCC) compared to homologous normal kidney tissue. Two-dimensional electrophoresis was used to separate and visualize HSP27, via immunostaining with anti-HSP27 antibody, in tumor and normal renal samples, obtained after surgery resection from patients with RCC. The mean number of protein species was 21 in RCC and 15 in normal tissues. Selected spots were in-gel digested with trypsin, extracted and analyzed by microcapillary liquid chromatography (LC) electrospray ionization tandem mass spectrometry to confirm HSP27 protein identity and reveal phosphorylation sites. Loss of phosphopeptides due to extensive plumbing and/or metal components in automated LC-systems was limited by manual loading of samples directly onto the LC system using a homemade pressure vessel. Mass spectrometry (MS) analysis revealed that in three of the HSP27 protein species phosphorylation occurred at Serine 15 and in five at Serine 82 in a different pattern. The phosphorylation of Serine 15 and 82 was also investigated by immunohistochemistry on tissue sections. The data obtained using anti-HSP27Serine82phos-antibody are consistent with MS results, while the variance between results achieved by anti-HSP27Serine15phos-antibody and by MS is probably due to the low specificity of the antibody. Knowledge of the diversity and modulation of HSP27 phosphorylation protein species might represent useful markers involved in the differentiation of RCC. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A combined proteomic and transcriptomic analysis of Staphylococcus aureus strain N315 was performed to study a sequenced strain at the system level. Total protein and membrane protein extracts were prepared and analyzed using various proteomic workflows including: 2-DE, SDS-PAGE combined with microcapillary LC-MALDI-MS/MS, and multidimensional liquid chromatography. The presence of a protein was then correlated with its respective transcript level from S. aureus cells grown under the same conditions. Gene-expression data revealed that 97% of the 2'596 ORFs were detected during the post-exponential phase. At the protein level, 23% of these ORFs (591 proteins) were identified. Correlation of the two datasets revealed that 42% of the identified proteins (248 proteins) were amongst the top 25% of genes with highest mRNA signal intensities, and 69% of the identified proteins (406 proteins) were amongst the top 50% with the highest mRNA signal intensities. The fact that the remaining 31% of proteins were not strongly expressed at the RNA level indicates either that some low-abundance proteins were identified or that some transcripts or proteins showed extended half-lives. The most abundant classes identified with the combined proteomic and transcriptomic approach involved energy production, translational activities and nucleotide transport, reflecting an active metabolism. The simultaneous large-scale analysis of transcriptomes and proteomes enables a global and holistic view of the S. aureus biology, allowing the parallel study of multiple active events in an organism.

The SWISS-2DPAGE database was established in 1993 and is maintained collaboratively by the Swiss Institute of Bioinformatics (SIB) and the Biomedical Proteomics Research Group (BPRG) of the Geneva University Hospital. During these years, SWISS-2DPAGE underwent constant modification and improvement. Current content includes about 4000 identified spots corresponding to 1200 different protein entries in 36 reference maps from human, mouse, Arabidopsis thaliana, Dictyostelium discoideum, Escherichia coli, Saccharomyces cerevisiae and Staphylococcus aureus origins. With a high level of annotation and integration with other relevant databases, SWISS-2DPAGE is a reference source in the proteomics world. Queries to SWISS-2DPAGE database currently reach 1000 hits per day.

In the present study, the effect of a high fat diet on the expression of proteins in insulin target tissues was analyzed using a proteomic approach. Gastrocnemius muscle, white and brown adipose tissue, and liver were taken from C57BL/6 mice either fed on a high-fat or a chow diet. Expression levels of approximately 10 000 polypeptides for all the four tissues were assessed by two-dimensional gel electrophoresis (2-DE). Computer-assisted image analysis allowed the detection of 50 significantly (p < 0.05) differentially expressed proteins between obese and lean mice. Interestingly, more than half of these proteins were detected in the brown adipose tissue. The differentially expressed proteins were identified by tandem mass spectrometry. Several stress and redox proteins were modulated in response to the high-fat diet. A key glycolytic enzyme was found to be downregulated in adipose tissues and muscle, suggesting that at elevated plasma fatty acid concentrations, fatty acids compete with glucose as an oxidative fuel source. Furthermore, in brown adipose tissue there were significant changes in mitochondrial enzymes involved in the Krebs tricarboxylic acid (TCA) cycle and in the respiratory chain in response to the high-fat diet. The brown adipose tissue is an energy-dissipating tissue. Our data suggest that the high-fat diet treated mice were increasing energy expenditure to defend against weight gain.

Only few biological markers are currently available for the routine diagnosis of brain damage-related disorders including cerebrovascular, dementia, and other neurodegenerative diseases. In this study, post-mortem cerebrospinal fluid samples were used as a model of massive brain insult to identify new markers potentially relevant for neurodegeneration. The protein pattern of this sample was compared to the one of cerebrospinal fluid from healthy subjects by two-dimensional gel electrophoresis. Using gel imaging, N-terminal microsequencing, mass spectrometry, and immunodetection techniques, we identified 13 differentially expressed proteins. Most of these proteins have been previously reported to be somehow associated with brain destruction or with the molecular mechanisms underlying certain neurodegenerative conditions. These data indicate that the identified proteins indeed represent potential biomarkers of brain damage. We recently showed that H-FABP, a protein highly homologous to E-FABP and A-FABP identified in this study, is a potential marker of Creutzfeldt-Jakob disease and stroke.

Approaches aimed at deciphering the proteome have illustrated the need for relatively complex and highly sensitive methodologies. The major elements of proteome analysis, such as powerful protein separation and enzymatic processing, mass spectrometry and dedicated bioinformatics have been assembled in the development of the molecular scanner. This highly flexible and data-rich approach has combined the power of electrophoretic protein separation, the simultaneous digestion and transfer of proteins through an enzymatic membrane, the immediate use of the MALDI mass spectrometer to scan a collecting membrane, and the development of dedicated bioinformatics tools to perform protein identification and molecular imaging of the proteome. Clinical applications of the molecular scanner have also started to be developed for disease diagnosis in biological material.

Protein copy number is distributed from 7 to 8 orders of magnitude in cells and probably up to 12 orders of magnitude in plasma. Classical silver-stained two-dimensional electrophoresis (2-DE) can only display up to four orders of magnitude. This is a major drawback since it is assumed that most of the regulatory proteins are low-abundance gene products. It is thus clear that the separation of low copy number proteins in amounts sufficient for postseparation analysis is an important issue in proteome studies to complete the comprehensive description of the proteome of any given cell type. The visualization of a polypeptide on a 2-DE gel will depend on the copy number, on the quantity loaded onto the gel and on the method of detection. As the amount of protein that can be loaded onto a gel is limited, one efficient solution is to fractionate the sample prior to 2-DE analysis. Several approaches exist including subcellular fractionation, affinity purification and chromatographic and electrophoretic protein prefractionation. The chromatographic step adds a new dimension in the protein separation using specific protein properties. It allows proteins to be adsorbed to a surface and eluted differentially under certain conditions. This review article presents studies combining chromatography-based methods to 2-DE analysis and draws general conclusions on this strategy.

Protein identification using automated data-dependent tandem mass spectrometry (MS/MS) is now a standard procedure. However, in many cases data-dependent acquisition becomes redundant acquisition as many different peptides from the same protein are fragmented, whilst only a few are needed for unambiguous identification. To increase the quality of information but decrease the amount of information, a nonredundant MS (nrMS) strategy has been developed. With nrMS, data analysis is an integral part of the overall MS acquisition and analysis, and not an endpoint as typically performed. In this nrMS workflow a matrix assisted laser desorption/ionization-time of flight-time of flight (MALDI-TOF/TOF) instrument is used. MS and restricted MS/MS data are searched and identified proteins are used to generate an "exclusion list", after in silico digestion. Peptide fragmentation is then restricted to only the most intense ions not present in the exclusion list. This process is repeated until all peaks are accounted for or the sample is consumed. Compared to nanoLC-MS/MS, nrMS yielded similar results for the analysis of six pooled two-dimensional electrophoresis (2-DE) spots. In comparison to standard data-dependent MALDI-MS/MS for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel band analysis, nrMS dramatically increased the number of identified proteins. It was also found that this new workflow significantly increased sequence coverage by identifying unexpected peptides, which can result from post-translational modifications.

Apolipoprotein E (apo E) is a normal component of several classes of plasma lipoproteins. Apo E phenotypes are closely related to total cholesterol, low density lipoprotein (LDL)-cholesterol and apo B concentration. The apo E 2/2 phenotype is related to the type III hyperlipoproteinemia due to the defective binding of apo E-2 to the hepatic receptors. The apo E 4/4 phenotype has been reported to be present in most elderly people suffering from the Alzheimer disease, and is associated with increased risk of coronary heart disease and Creutzfeld-Jakob disease. Therefore, apo E phenotyping is essential. The method described here uses a precast immobilized pH gradient, avoids time-consuming separation of lipoproteins from plasma, needs no pretreatment with neuraminidase and involves highly sensitive enhanced chemiluminescence for visualization. Therefore it has many advantages over previously published methods.

The synthesis and application of two new alkylating reagents, N-tert-butyl-2-iodoacetamide (N-t-butyliodoacetamide) and 2-iodo-N-phenylacetamide (iodoacetanilide), are described. N-t-Butyliodoacetamide and iodoacetanilide were synthesised to purity in their d(0)-light and in their respective d(9)- and d(5)-heavy forms. The newly synthesised reagents are covalently bound to peptides containing cysteines via an alkylation reaction. The mass differences of 5 and 9 Da avoid possible problems of overlapping isotope distribution. For each alkylated cysteine a peptide mass increases, respectively, by a multiple of 113 and 133 Da for the d(0)-light form of N-t-butyliodoacetamide and iodoacetanilide. These reagents can therefore replace common alkylating reagents in existing proteomics-based applications. Alkylated peptides increase in mass in the same mass range as amino acids and remain suitable for tandem mass spectrometry (MS/MS) data acquisition and analysis. The compounds are simple to use and derivatisation is based on widely applied alkylating procedures. Preliminary results show that these reagents can be applied for both protein quantitation and identification by peptide mass finger printing and/or MS/MS techniques. Using these chemicals and the suggested workflow enables the quantitative analysis of the whole protein sample and realises access to peptides that may contain potential post-translational modifications. Other approaches that incorporate a matrix-assisted laser desorption/ionisation (MALDI) interface prior to MS can take advantage of these chemicals, such as the molecular scanner.

Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.

The diagnosis of Alzheimer's disease (AD), the most common form of dementia in the general population, usually relies upon the presence of typical clinical features and structural changes on brain magnetic resonance imaging. Over the last decade, a number of biological abnormalities have been reported in the cerebrospinal fluid (CSF) of AD patients, in particular altered levels of the tau protein and the 1-42 fragment of the amyloid precursor protein. These, however, have not yet proved sensitive and specific enough to be included in the diagnostic criteria for AD, leaving plenty of room for the search of novel biomarkers. The present study describes the analysis of CSF polypeptides by a protein-chip array technology called surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Using this approach, we detected statistically significant quantitative differences (p < 0.05) regarding four overexpressed and one underexpressed polypeptides in the CSF of AD patients as compared to healthy controls. Four of them were further purified by strong anionic exchange chromatography (SAX) and identified by MS analysis as cystatin C, two beta-2-microglobulin isoforms, an unknown 7.7 kDa polypeptide, and a 4.8 kDa VGF polypeptide. The combination of the five polypeptides for the diagnosis of AD allowed to classified six AD patients out of the nine included in this study and all the ten controls, which means in this small cohort that the specificity and sensitivity are 100% and 66%, respectively. This study, based on the protein-chip array technology, demonstrates the presence in the CSF of novel potential biomarkers for AD, which may be used for the diagnosis and perhaps the assessment of the severity and progression of the disease.

The recent occurrence of the new variant of Creutzfeldt-Jakob disease (CJD), probably transmitted to humans by cattle affected by the bovine form of spongiform encephalopathy, has generated renewed interest in the clinical issues related to human spongiform encephalopathies. Using the current set of diagnostic tools, these rare but devastating conditions may be difficult to diagnose with accuracy before death. The objective of the present communication is to describe the discovery of a potential cerebrospinal fluid (CSF) and plasmatic marker of human transmissible spongiform encephalopathies. A preliminary two-dimensional electrophoresis approach highlighted a potential neurodegenerative disorder marker called the fatty acid binding protein, FABP. Its heart form, H-FABP, was investigated in a small group of CJD affected patients (n = 8 ) by an immunoassay approach. The amount of FABP appeared to be significantly (p< or = 0.05) increased in all tested samples. H-FABP detection could therefore be helpful as a blood screening test for a pre-mortem diagnosis of the disease and also to prevent the risk of iatrogenic transmission of CJD through blood transfusion.

For some years now, scientists have been spending a lot of effort in developing methods to analyse and compare complex protein samples. One of the goals ol such global analyses of what is known as proteomes is to discover specific protein markers - or fingerprints ol protein markers - from various types of affected biological samples. Considering the battery of technologies currently available, mass spectrometry (MS) constitutes an essential tool in proteomics. We describe here the type of MS instrumentation that is currently dedicated to proteomics research. We also describe the major experimental workflows that are typically used in proteomics today, with a focus on those incorporating MS as a major analysis tool.

Peroxisome proliferator-activated receptors (PPARs) participate in the molecular mechanism of pathologies with altered lipid homeostasis such as type 2 diabetes or obesity. The insulin sensitizer drug, rosiglitazone, has been shown to bind and activate PPAR-gamma1 in adipocytes and PPAR-gamma2 in hepatocytes. The identification of new molecular targets associated with fatty acid oxidation and PPAR-gamma nuclear receptor regulation in insulin resistance tissues is a key research goal. In the present study, we have used a proteomic approach to identify such targets. Lean and obese C57 Bl/6J lep/lep mice were given BRL49653, rosiglitazone, 10 mg/kg diet, by dietary admixture for 7 days. Rosiglitazone normalized the impaired glucose tolerance and dyslipidemia in lep/lep mice but had no significant effect in the lean mice. Samples of liver, white and brown adipose tissue, and muscle proteins were obtained and 100 microg of proteins was arrayed by two-dimensional gel electrophoresis. Thirty-four polypeptides were differentially expressed (p < 0.05) between lep/lep and lean mice and eleven were significantly (p < 0.05) modulated by rosiglitazone treatment of the obese mice. None of the proteins was modulated by rosiglitazone treatment of the lean mice. The identity of these differentially expressed proteins was made using tandem mass spectrometric analysis and revealed components of fatty acid and carbohydrate metabolism as well as proteins with unknown function.

The MELANIE expert system recognized and blindly separated normal and cirrhotic liver or different cancer biopsy protein patterns. The discovery of a new lipoprotein associated protein highlighted the need for linkages between protein map databases and the human genome databases. The results described in this manuscript clearly demonstrate that some diseases associated with toxic compounds or modifications of the human genome are adequately diagnosed by expert systems which automatically analyze protein maps. The potential of this combination of biotechnology (protein mapping) and computer analysis is the development of a 'molecular scanner' in medicine. The massive amount of information analyzed and stored requires new strategies including centralized protein map databases and image transmission over networks. The increasing knowledge in protein expression and regulation will amplify the impact of the human genome project in medicine and biology. >

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry has become a valuable tool in proteomics. With the increasing acquisition rate of mass spectrometers, one of the major issues is the development of accurate, efficient and automatic peptide mass fingerprinting (PMF) identification tools. Current tools are mostly based on counting the number of experimental peptide masses matching with theoretical masses. Almost all of them use additional criteria such as isoelectric point, molecular weight, PTMs, taxonomy or enzymatic cleavage rules to enhance prediction performance. However, these identification tools seldom use peak intensities as parameter as there is currently no model predicting the intensities based on the physicochemical properties of peptides. In this work, we used standard datamining methods such as classification and regression methods to find correlations between peak intensities and the properties of the peptides composing a PMF spectrum. These methods were applied on a dataset comprising a series of PMF experiments involving 157 proteins. We found that the C4.5 method gave the more informative results for the classification task (prediction of the presence or absence of a peptide in a spectra) and M5' for the regression methods (prediction of the normalized intensity of a peptide peak). The C4.5 result correctly classified 88% of the theoretical peaks; whereas the M5' peak intensities had a correlation coefficient of 0.6743 with the experimental peak intensities. These methods enabled us to obtain decision and model trees that can be directly used for prediction and identification of PMF results. The work performed permitted to lay the foundations of a method to analyze factors influencing the peak intensity of PMF spectra. A simple extension of this analysis could lead to improve the accuracy of the results by using a larger dataset. Additional peptide characteristics or even PMF experimental parameters can also be taken into account in the datamining process to analyze their influence on the peak intensity. Furthermore, this datamining approach can certainly be extended to the tandem mass spectrometry domain or other mass spectrometry derived methods.

The development of high throughput utilities to identify proteins is a major challenge in present research in the field of proteomics. One such utility, the molecular scanner, uses proteins separated by two-dimensional polyacrylamide gel electrophoresis that are digested in the gel and during transfer onto a collecting membrane. After adding a matrix, the membrane is inserted into a matrix-assisted laser desorption/ionization-time of flight mass spectrometer and a peptide mass fingerprint (PMF) is measured for every scanned site. Since the spacing between scanned sites is much smaller than the size of the most abundant protein spots, there is a certain redundancy in the data that was used in an earlier experiment with Escherichia coli [1] to improve mass calibration and PMF identification results. It was observed that the signal intensity of a peptide mass as a function of the position on the membrane showed similar patterns if peptides stemmed from the same protein. Taking account of these similarities a clustering algorithm was used to find lists of experimental masses with similar intensity distributions, which provided clearer identification of the corresponding proteins. Here, these methods are applied to a human plasma scan, where proteins were highly modified and less separated. The presence of very abundant proteins like albumin and immunoglobulins added another difficulty. The calibration of the initial PMFs was not satisfactory and masses had to be recalibrated. After discarding chemical noise, the membrane was partitioned into regions and for each region protein identification was carried out separately. A new scoring method was used, where the PMF score was multiplied by a factor that measures the similarity of matching peptides. This method proved to be more robust than the method developed in [1] if the region where a protein was found had an extended, nonspherical shape and strong overlap with regions of other proteins. Many proteins annotated on the SWISS-2D PAGE human plasma master gel could be clearly identified and many interesting properties were observed.

The notion of a “plurifunctional” nucleolus is now well established. However, molecular mechanisms underlying the biological processes occurring within this nuclear domain remain only partially understood. As a first step in elucidating these mechanisms we have carried out a proteomic analysis to draw up a list of proteins present within nucleoli of HeLa cells. This analysis allowed the identification of 213 different nucleolar proteins. This catalog complements that of the 271 proteins obtained recently by others, giving a total of ∼350 different nucleolar proteins. Functional classification of these proteins allowed outlining several biological processes taking place within nucleoli. Bioinformatic analyses permitted the assignment of hypothetical functions for 43 proteins for which no functional information is available. Notably, a role in ribosome biogenesis was proposed for 31 proteins. More generally, this functional classification reinforces the plurifunctional nature of nucleoli and provides convincing evidence that nucleoli may play a central role in the control of gene expression. Finally, this analysis supports the recent demonstration of a coupling of transcription and translation in higher eukaryotes.

The insulin sensitizer drug, rosiglitazone, has been shown to have a protective effect on pancreatic islet cell structure and function in animal models of type 2 diabetes. The identification of new molecular targets associated both with islet cell dysfunction and protection is a crucial research goal. In the present study, a proteomics approach has been used to identify such targets. Obese C57Bl/6J lep/lep mice and lean littermates were given the insulin sensitizer drug BRL49653, rosiglitazone. It normalized the impaired glucose tolerance in lep/lep mice but had no significant effect on glucose tolerance in the lean mice. Pancreatic islet polypeptides were arrayed by a two-dimensional gel electrophoresis system that separated more than 2500 individual spots. Three overexpressed and six underexpressed proteins were significant (p < 0.05) between lep/lep and lean mice, and four were modulated significantly (p < 0.05) by the rosiglitazone treatment of the obese mice. The identity of these differentially expressed proteins was made using mass spectrometric analysis and provided evidence that differential expression of actin-binding proteins may be an important aspect of defective islet function. Rosiglitazone increased carboxypeptidase B expression in both lep/lep and normal mice suggesting that this might be an independent effect of rosiglitazone that contributes to improved insulin processing.

Protein identification is becoming a complement to the available fully sequenced genomes. To meet the challenge, newly developed techniques for high throughput protein identification using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and peptide mass fingerprint are needed. Two years ago, a parallel protein digestion process was proposed. It provided a collecting polyvinylidene difluoride (PVDF) membrane able to be scanned by MALDI. Acquired data were used to recreate a virtual multidimensional image. Voltage used during this protein electroblotting technique was an unusual square wave alternative voltage (SWAV). The goal of the current study is to evaluate quantitatively the efficiency of the SWAV compared with a classical electroblot process on intact proteins. The effect of the pulsed electric field and the buffer composition were compared to a standard continuous transblotting process defined as the gold standard. Combination of the pulsed asymmetric electric field with 3-(cyclohexylamino)-1-propane-sulfonique acid (CAPS) buffers showed an average 65% increase of protein recovery. Moreover, a strongest effect is observed for high M(r) proteins. In conclusion, the present study highlighted a positive influence of the "shaking" effect of the asymmetric alternative voltage on gel protein extraction.

The complexity of nature is tremendous, particularly at the epigenetic level. Proteomic studies must therefore complement genomic discoveries to better understand biological processes. Because of the very large number of modified proteins and their great variability in physico-chemical properties, no single method can be used to analyze all of them. Mass spectrometry has demonstrated its superior ability to rapidly identify and partially characterize numerous proteins in low abundance and has become a central element in most proteomic projects. Studies of protein function are necessary to understand biological pathways and this is being tackled using several approaches such as two hybrid systems, phage technology or affinity methods. Finally, mathematical and bioinformatic developments will be essential to study nature's complex systems.

The solubilized proteins of purified CD19(+) (B), CD8(+) (T) as well as CD4(+) (T) lymphocytes were separated by high resolution two-dimensional polyacrylamide gel electrophoresis, and the gels were analyzed using Melanie 3.0. Nine gels were studied, three for each lymphocyte population. After image analysis, 1411 +/- 73 spots (mean + SD) were detected. The protein pattern of B lymphocytes segregated from the one of T lymphocytes by ascendant heuristic clustering analysis. In addition, computer analysis separated CD8(+) from CD4(+) lymphocytes. When a search was performed in order to detect subsets of specific spots (presence vs. absence), a group of three spots, detected in the area of the protein maps corresponding to isoelectric point (pI) of 5.2 to 5.4 and molecular weight (M(r)) of 50 to 51 kDa, were found in both CD8(+) and CD4(+) cells, but not in CD19(+) cells. Mass spectrometry analysis revealed that these spots were associated with several proteins such as vimentin, tubulin, desmin and cytokeratin. Two spots, located in the area of the gel corresponding to pI of about 5.0 and a M(r) of 30 kDa, appeared as CD8(+) cell associated. Mass spectrometry analysis showed that the two spots were related to the same non-identified protein. Moreover internal peptides sequences matched with two human expressed sequence tags: gi/9759776, gi/12798420. No spots were found as only B cell associated.

In 1993, we reported the presence of an IgM-associated peptide (M(r) 44 kDa; pI 5.45) in all immunoglobulin M (IgM) fractions purified from plasma/serum by various methods. This peptide was absent in Ig fractions of non-IgM isotypes. The N-terminal sequence was determined as being APPSGVRLVGGLH. To gain insight into the nature of this peptide, we further analyzed, using modern proteomic tools, the IgM-associated peptide isolated from cryoglobulins. Mass spectrometry revealed three peptides of different masses: 2203.13 (ELGCGAASGTPSGILYEPPAEK), 1564.83 (KPIWLSQMSCSGR), and 1544.77 (EATLQDCPSGPWGK). Theses sequences together with the already known N-terminal sequence allowed us to identity the IgM-associated peptide as Sp alpha (O43866 in TrEMBL database; CD5 antigen-like). Sp alpha is a member of the scavenger receptor cysteine-rich superfamily of proteins. This family includes the T-and B-cell antigens CD5 and CD6, and several of its members influence immune cell fate. Our finding may have important implications in the understanding of the homeostasis of IgM antibodies.

Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments.

To study the clonal events occurring during ontogeny of the humoral immune system, we evaluated plasma immunoglobulin (Ig) production in term newborns and young children by high-resolution two-dimensional gel electrophoresis. The clonality pattern of Ig light (L) chains from healthy newborns (n = 19) was similar to that observed on protein maps of their mothers or of normal adults (n > 100), that is, rare distinguishable small spots in a cloud-like large band of indiscrete Ig L chain spots (polyclonal pattern; maternal Igs). Analysis of plasma samples obtained from infants between 1 month and 5 years of age (n = 55) revealed discrete but evident alterations of the clonality of Ig production. Between the 2nd and 4th months of life, transient attenuation of the "polyclonal background" was observed in association with the appearance of an increasing number of well-resolved Ig L chain spots (corresponding to plasma Ig concentrations between 0.5 and 1 g/L per spot). This "restricted" clonal pattern was progressively less apparent on protein maps of infants older than 2 year and evolved towards a "normal" adult polyclonal pattern at the age of 5. These results suggest that the development of the B-cell clones is heterogeneous, either through limited outgrowth of precursor cells or through selective antigenic pressures.

This publication updates the reference human liver protein map. By microsequencing, 27 spots or 34 polypeptide chains were identified. The most abundant polypeptides detected on the silver stained liver map were key elements in major hepatic biochemical pathways. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing the 1993 reference human liver SWISS-2DPAGE database. SWISS-2DPAGE and the SWISS-PROT protein sequence databases are closely linked together through the use of common accession numbers.

Renal cell carcinoma (RCC) originates in the renal cortex. It accounts for 2 —3 percent of all cancers occurring in adults and it is characterised by lack of early clinical manifestations, un- predictable outcome, and absence of effective treatment modalities except early surgery. RCC com- prises a heterogeneous group of tumours with various molecular and cytogenetic abnormalities and different histological features as cell types and tumour architecture. Molecular genetic and proteo- mic tools led to the discovery of potential diagnostic prognostic and therapeutic biomarkers of RCC. In this review we discuss recent developments in understanding genotype-phenotype rela- tionships, with attention to manganese superoxide dismutase, a mitochondrial enzyme related to the redox cycle which affects various regulatory functions of cells. The expression of this protein has been evaluated in numerous human tumour types including RCC, and post-translational modifica- tions are being investigated. Keywords. Two-dimensional electrophoresis / Renal cell carcinoma / Manganese superoxide dis- mutase / Post-transitional modifications / Review Abbreviations. IFN, interferon; IL, interleukin; Mn-SOD, manganese superoxide dismutase; RCC, renal cell carcinoma

Pallidal hyperintensity at magnetic resonance imaging (MRI) correlates to blood manganese (Mn) levels and parkinsonian signs in patients with cirrhosis. Similarly, metabolite changes in the basal ganglia (BG) at proton spectroscopy are related to these neurological signs. The evolution of these abnormalities after liver transplantation (OLT) is incompletely described. We evaluated 14 unselected consecutive patients with cirrhosis (minimal hepatic encephalopathy [HE] n=8, no HE n=6) before and 4 months after successful OLT for the evolution of parkinsonism using a validated scale (the United Parkinson's Disease Rating Scale, or UPDRS). Pallidal intensity at MRI, spectroscopic changes in the BG at magnetic resonance spectroscopy (MRS), and whole blood manganese concentrations were measured. After OLT in patients with preoperative minimal HE, the UPDRS scores improved, but mild parkinsonism persisted (16.1+/-3.6 to 6.2+/-4.8, P

Objective: To determine the usefulness of the 14-3-3 test in patients with dementia of various causes. Background: Recent reports have suggested that the detection of the 14-3-3 protein in the CSF of patients with Creutzfeldt–Jakob disease is a highly sensitive and specific marker of the disease that might be used as a diagnostic criterion. We examined the validity of this test when applied to a cohort of unselected patients prospectively examined for an ongoing dementing process. Methods: One hundred patients underwent an extensive neurologic examination for dementia, including a CSF 14-3-3 protein immunoblotting assay. Final clinical diagnoses were compared with the qualitative results of the test, and statistical measures of test validity were carried out. Results: We found a positive test in 14 of 100 patients, only two of whom had definite Creutzfeldt–Jakob disease. Positive results were found in patients with various degenerative dementias, including AD (4), frontotemporal dementia (2), and dementia with Lewy body (1), and in patients with vascular dementia (1), carcinomatous meningitis (1), and anoxic encephalopathy (1). In two other positive patients, the dementia could not be confidently classified. Sensitivity, specificity, and negative predictive value were fairly good, but positive predictive value was poor. Similar results were found independently of the disease duration. There was no correlation between intensity nor pattern of the 14-3-3 protein expression and diagnosis. Conclusions: The 14-3-3 test is not valid for discriminating between Creutzfeldt–Jakob disease and non-Creutzfeldt–Jakob disease in unselected patients with dementia. Positive results are found in various degenerative and secondary, prion-unrelated dementias.

In this work we extended the study of genes controlling the formation of specific differentiation structures called “domes” formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the β-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC β-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC β-subunit and the rat8 proteins, and is under the negative control of maspin.

Proteomics-based approaches, which examine the expressed proteins of a tissue or cell type, complement the genome initiatives and are increasingly being used to address biomedical questions. Proteins are the main functional output, and the genetic code cannot always indicate which proteins are expressed, in what quantity, and in what form. For example, post-translational modifications of proteins, such as phosphorylation or glycosylation, are very important in determining protein function. Similarly, the effects of environmental factors or multigenic processes such as ageing or disease cannot be assessed simply by examination of the genome alone. This review describes the underlying technology and illustrates several areas of biomedical research, ranging from pathogenesis of neurological disorders to drug and vaccine design, in which potential clinical applications are being explored.

SWISS-2DPAGE (http://www.expasy.ch/ch2d/ ) is an annotated two-dimensional polyacrylamide gel electro-phoresis (2-DE) database established in 1993. The current release contains 24 reference maps from human and mouse biological samples, as well as from Saccharomyces cerevisiae, Escherichia coli and Dictyostelium discoideum origin. These reference maps have now 2824 identified spots, corresponding to 614 separate protein entries in the database, in addition to virtual entries for each SWISS-PROT sequence or any user-entered amino acids sequence. Last year improvements in the SWISS-2DPAGE database are as follows: three new maps have been created and several others have been updated; cross-references to newly built federated 2-DE databases have been added; new functions to access the data have been provided through the ExPASy proteomics server.