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PubMed2005

Report of the roundtable discussion organised by the Swiss Proteomics Society (SPS), Bern, 8th December 2004.

How close are we to using proteomics tools in the every day practice of physicians? What are the socio-economical issues our health care system may face with the advent of biomarkers for early diagnosis? How to get the specialists from the various disciplines integrated in proteomics to establish a common understanding of the clinical issues and develop the necessary standards (methods, biochemicals and IT)? These were the kind of questions a panel of specialists tried to answer during the roundtable discussion that took place in Bern during the Swiss Proteomics Society 2004 congress.

Automation↗
PubMed2006

A functional proteomics approach for the detection of nuclear proteins based on derepressed importin alpha.

The identification of functional proteomes is a major challenge in proteomic research. Here we describe a method for the detection and isolation of nuclear (localization sequence containing) proteins using a derepressed import receptor (DIRE) as a synthetic antibody. We demonstrate that the DIRE method specifically detects nuclear localization sequence containing proteins. Application to activation of primary T-lymphocytes exemplifies the potential use of DIRE for comparative proteomics and for diagnostics.

Amino Acid Sequence↗
PubMed2006

Proteome analysis of salt stress response in the cyanobacterium Synechocystis sp. strain PCC 6803.

In the present study, changes in protein synthesis patterns after salt shock visualized by 35S-methionine labeling and the changed protein composition in salt-acclimated cells of the cyanobacterium Synechocystis sp. strain PCC 6803 were analyzed by a combination of 2-DE for protein separation and PMF for protein identification. As a basis for the differential analysis, a proteome map with 500 identified protein spots comprising 337 different protein species was established. Fifty-five proteins were found, which are induced by salt shock or accumulated after long-term salt acclimation. Some of the proteins are salt stress-specific, such as enzymes involved in the synthesis of the compatible solute glucosylglycerol, while most of them are involved in general stress acclimation. Particularly, heat-shock proteins and proteins acting against lesions by reactive oxygen species were found. Moreover, changes in enzymes involved in basic carbohydrate metabolism were detected. The dynamic of the proteome of salt-stressed Synechocystis cells was compared to previous data concerning transcriptome analysis revealing that 89% of the proteins induced shortly after salt shock were also found to be induced at the RNA level. However, 42% of the stably up-regulated proteins in salt-acclimated cells were not detected previously using DNA microarrays. The comparison of transcriptomic and proteomic analyses shows the significance of post-transcriptional regulatory mechanisms in acclimation of Synechocystis to high salt concentrations.

Bacterial Proteins↗
PubMed2006

Antibodies for proteomic research: comparison of traditional immunization with recombinant antibody technology.

Antibodies play a pivotal role in studying the expression and function of proteins. Proteomics studies require the generation of specific and high-affinity antibodies against large numbers of proteins. While traditional animal-based antibody generation is laborious, difficult to automate, and therefore less suited to keep up with the requirements of proteomics research, the use of recombinant in vitro antibody technology might offer a solution to this problem. However, it has not been demonstrated yet that such antibodies are at least as useful as conventional antibodies for typical proteomics applications. Here we generated novel recombinant Fab antibody fragments from the naïve HuCAL GOLD library against a number of targets derived from a mouse cDNA library. We compared these antibodies with polyclonal antisera produced against the same targets and show that these recombinant antibodies are useful reagents for typical applications like Western blotting or immunohistochemistry.

Animals↗
PubMed2006

Construction of quantitative proteome reference maps of mouse spleen and lymph node based on two-dimensional gel electrophoresis.

Quantitative features of the proteome are extremely useful for studying cellular processes at a molecular level. In this study, we attempted to construct quantitative reference proteome maps of the mouse spleen and lymph node based on 2-DE followed by protein identification using MS. We analyzed more than 1000 spots on the 2-DE images and consequently were able to determine that 919 spots were derived from 328 different genes. To obtain statistically reliable information of the protein levels from these 2-DE images, we measured the volumes of the respective spots on 2-DE images obtained by four to six independent experimental runs. These measurements were used to calculate the variability of the volumes of the respective spots on 2-DE following subcellular fractionation, which enabled us to discriminate differentially produced proteins from those within the range of intrinsic variability. More importantly, while the 2-DE data have been traditionally collected in a gel image-based manner, the resultant quantitative 2-DE data could be analyzed using the same procedure as that for mRNA expression profiles. This greatly assists in bridging the gap between the analyses of transcriptomes and proteomes and enables the integration of this data on the same informational platform.

Amino Acid Sequence↗
PubMed2005

Further steps in standardisation. Report of the second annual Proteomics Standards Initiative Spring Workshop (Siena, Italy 17-20th April 2005).

The spring workshop of the HUPO-PSI convened in Siena to further progress the data standards which are already making an impact on data exchange and deposition in the field of proteomics. Separate work groups pushed forward existing XML standards for the exchange of Molecular Interaction data (PSI-MI, MIF) and Mass Spectrometry data (PSI-MS, mzData) whilst significant progress was made on PSI-MS' mzIdent, which will allow the capture of data from analytical tools such as peak list search engines. A new focus for PSI (GPS, gel electrophoresis) was explored; as was the need for a common representation of protein modifications by all workers in the field of proteomics and beyond. All these efforts are contextualised by the work of the General Proteomics Standards workgroup; which in addition to the MIAPE reporting guidelines, is continually evolving an object model (PSI-OM) from which will be derived the general standard XML format for exchanging data between researchers, and for submission to repositories or journals.

Mass Spectrometry↗
PubMed2006

Proteomic analysis of anaplastic lymphoma cell lines: identification of potential tumour markers.

Anaplastic large-cell lymphomas (ALCL) are high grade lymphomas of T or null phenotype often associated with the t(2;5) translocation leading to the expression of a chimeric protein consisting of the N-terminal portion of nucleophosmin (NPM) and the intracellular domain of the anaplastic lymphoma kinase (ALK). Although ALCL are recognized as distinct clinical, biological and cytogenetic entities, heterogeneities persist in this group of tumours, which exhibit a broad spectrum of morphological features. Particularly, the common type tumour consisting in large cells contrast with the small cell variant that is sometimes associated with a leukemic phase. The ALK-negative ALCL is often associated with a poor prognosis. Here, we investigated the proteome of these subtypes of tumours using patient-derived cell lines. We compared the proteome of the cytosolic fraction of NPM-ALK-positive versus NPM-ALK-negative cells on one hand, and the proteome of common cell type versus small cell variant on the other hand. The identification of a set of proteins differentially expressed in the subtypes of ALCL points to new diagnosis/prognosis markers. This study also provides interesting information on the molecular mechanisms responsible for the different subtypes of ALCL.

Anaplastic Lymphoma Kinase↗
PubMed2006

Modeling a whole organ using proteomics: the avian bursa of Fabricius.

While advances in proteomics have improved proteome coverage and enhanced biological modeling, modeling function in multicellular organisms requires understanding how cells interact. Here we used the chicken bursa of Fabricius, a common experimental system for B cell function, to model organ function from proteomics data. The bursa has two major functional cell types: B cells and the supporting stromal cells. We used differential detergent fractionation-multidimensional protein identification technology (DDF-MudPIT) to identify 5198 proteins from all cellular compartments. Of these, 1753 were B cell specific, 1972 were stroma specific and 1473 were shared between the two. By modeling programmed cell death (PCD), cell differentiation and proliferation, and transcriptional activation, we have improved functional annotation of chicken proteins and placed chicken-specific death receptors into the PCD process using phylogenetics. We have identified 114 transcription factors (TFs); 42 of the bursal B cell TFs have not been reported before in any B cells. We have also improved the structural annotation of a newly sequenced genome by confirming the in vivo expression of 4006 "predicted", and 6623 ab initio, ORFs. Finally, we have developed a novel method for facilitating structural annotation, "expressed peptide sequence tags" (ePSTs) and demonstrate its utility by identifying 521 potential novel proteins from the chicken "unassigned chromosome".

Amino Acid Sequence↗
PubMed2006

Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels.

In an effort to contribute to a better understanding of the hepatic toxicity of the ubiquitous environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a comprehensive quantitative proteome analysis was performed on 5L rat hepatoma cells exposed to 1 nM TCDD for 8 h. Changes in the abundances of individual protein species in TCDD-treated cells as compared to untreated cells were analysed using the nongel-based isotope-coded protein label (ICPL) method [Schmidt, A., Kellermann, J., Lottspeich, F., Proteomics 2005, 5, 4-15]. 89 proteins were identified as up- or down-regulated by TCDD. For the majority of the altered proteins, an impact of TCDD on their abundance had not been known before. Due to the physicochemical properties or the translational regulation of a large number of the affected proteins, their alteration would have escaped detection by gel-based methods for proteome analysis and by standard mRNA expression profiling, respectively. The identified proteins with TCDD-altered abundance include several proteins implicated in cell cycle regulation, growth factor signalling and the control of apoptosis. The results thus provide new starting-points for the investigation of specific aspects of the toxicity and carcinogenicity of dioxin in liver.

Animals↗
PubMed2006

Quantitative proteomic profiling of pancreatic cancer juice.

Pancreatic juice is an exceptionally rich source of cancer-specific proteins shed from cancerous ductal cells into the pancreatic juice. Quantitative proteomic analysis of the proteins specific to pancreatic cancer juice has not previously been reported. We used isotope-code affinity tag (ICAT) technology and MS/MS to perform quantitative protein profiling of pancreatic juice from pancreatic cancer patients and normal controls. ICAT technology coupled with MS/MS allows the systematic study of the proteome and measures the protein abundance in pancreatic juice with the potential for development of biomarkers. A total of 105 proteins were identified and quantified in the pancreatic juice from a pancreatic cancer patient, of which 30 proteins showed abundance changes of at least twofold in pancreatic cancer juice compared to normal controls. Many of these proteins have been externally validated. This is the first comprehensive study of the pancreatic juice proteome by quantitative global protein profiling, and the study reveals numerous proteins that are shown for the first time to be associated with pancreatic cancer, providing candidates for diagnostic biomarkers. One of the identified proteins, insulin-like growth factor binding protein-2 was further validated by Western blotting to be elevated in pancreatic cancer juice and overexpressed in pancreatic cancer tissue.

Amino Acid Sequence↗
PubMed2006

Comparison of extraction procedures for proteome analysis of Streptococcus pneumoniae and a basic reference map.

Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.

Amino Acid Sequence↗
PubMed2006

Proteomic response of Schizosaccharomyces pombe to static and oscillating extremely low-frequency electromagnetic fields.

There is considerable public concern regarding the health effects of exposure to low-frequency electromagnetic fields. In addition, the association between exposure and disease incidence or the possible biological effects of exposure are unclear. Using 2D-DIGE and MS in a blind study, we have investigated the effects of static and oscillating extremely low-frequency electromagnetic fields (ELF EMFs) on the proteomes of wild type Schizosaccharomyces pombe and a Sty1p deletion mutant which displays increased sensitivity to a variety of cellular stresses. Whilst this study identifies a number of protein isoforms that display significant differential expression across experimental conditions, there was no correlation between their patterns of expression and the ELF EMF exposure regimen. We conclude that there are no significant effects of either static or oscillating EMF on the yeast proteome at the sensitivity afforded by 2D-DIGE. We hypothesise that the proteins identified must be sensitive to subtle changes in culture and/or handling conditions, and that the identification of these proteins in other proteomic studies should be treated with some caution when the results of such studies are interpreted in a biological context.

Electromagnetic Fields↗
PubMed2006

Proteome analysis of Schizosaccharomyces pombe by two-dimensional gel electrophoresis and mass spectrometry.

The fission yeast Schizosaccharomyces pombe (S. pombe) is a unicellular eukaryote and contains many genes and regulatory mechanisms that are close to those of mammals. In this study, we performed a global proteomic analysis of the fission yeast S. pombe wild type h(-S) L 972 proteome. More than 1,500 protein spots were visualized on silver stained 2-D gels in the 3-10 pI range with a high resolution and high reproducibility. Protein identification was carried out by MALDI-TOF-MS and/or nanoLC-MS/MS. Advantage of the complementarity of these two MS approaches was used to enhance the identification quality. So far, 364 proteins (representing 157 different proteins) have been identified. We report here the identification of 117 new proteins on our 2-D reference map of this yeast compared to the first reference map. Of these identified proteins, 40.1% were involved in metabolism. The present work provides a very useful tool for all studies relying on S. pombe as a model organism and is a considerable complement to the first reference map of S. pombe published recently by Sun and coworkers (Sun, N., Jang, J., Lee, S., Kim, S. et al.., Proteomics 2005, 5, 1574-1579).

Amino Acid Sequence↗
PubMed2006

Plasticity demonstrated in the proteome of a parasitic nematode within the intestine of different host strains.

The soluble global proteome of adult nematode Heligmosomoides polygyrus (H. p.) bakeri, a hookworm laboratory model was compared for the first time in the intestines of a slow-responder mouse host strain (C57/BL10) that is known to support a primary parasite infection for many months, and rapid-responder mouse host (SWR) that is known to eliminate the nematode infection by week 6 postinfection. At week 4 postinfection, major adult nematode proteins selectively produced following establishment of infection in C57/BL10 hosts include several globin forms, calreticulin and a phosphatidylethanolamine-binding protein. The increased synthesis of forms of myosin, actin and troponin in the nematode living in the rapid-responder SWR host may relate to the attempted reorganisation or repair of the cytoskeleton and/or muscle layer in the host immune initiated, increased mucus production and smooth muscle activity within intestinal environment. Initial evidence suggests weakly antigenic forms of globins dominant in the cytosol of H. p. bakeri adults in the intestinal environment compared to their low production in a related free-living nematode. The demonstration of considerable plasticity within a parasitic nematode proteome provides a molecular basis for the previously observed phenotypic plasticity within different host environments. Proteome plasticity has relevance to the efficiency of future vaccine and drug therapy, and the continued failure of defined antigen vaccines in mammalian populations.

Amino Acid Sequence↗
PubMed2006

Gel-free analysis of the human brain proteome: application of liquid chromatography and mass spectrometry on biopsy and autopsy samples.

This paper reports on the findings of the Biomedical Research Institute, as one of the participants in the pilot study of the HUPO Brain Proteome Project. A biopsy and autopsy study sample derived from human brain was distributed among the participants for proteomic analysis. In our laboratory, attention was focused on protein identification using the bottom-up shotgun approach. Protein extracts derived from both samples were trypsinized and analyzed separately by 2-D LC and MS. In a complementary approach, the tryptic digests were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a stringent evaluation process, resulted in the confident identification of 209 proteins in the human brain samples under investigation.

Adult↗
PubMed2006

Profiling human brain proteome by multi-dimensional separations coupled with MS.

In our initial attempt to analyze the human brain proteome, we applied multi-dimensional protein separation and identification techniques using a combination of sample fractionation, 1-D SDS-PAGE, and MS analysis. The complexity of human brain proteome requires multiple fractionation strategies to extend the range and total number of proteins identified. According to the method of Klose (Methods Mol. Biol. 1999, 112, 67), proteins of the temporal lobe of human brain were fractionated into (i) cytoplasmic and nucleoplasmic, (ii) membrane and other structural, and (iii) DNA-binding proteins. Each fraction was then separated by SDS-PAGE, and the resulting gel line was cut into approximately 50 bands. After trypsin digestion, the resulting peptides from each band were analyzed by RP-LC/ESI-MS/MS using an LTQ spectrometer. The SEQUEST search program, which searched against the IPI database, was used for peptide sequence identification, and peptide sequences were validated by reversed sequence database search and filtered by the Protein Hit Score. Ultimately, 1533 proteins could be detected from the human brain. We classified the identified proteins according to their distribution on cellular components. Among these proteins, 24% were membrane proteins. Our results show that the multiple separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.

Brain↗
PubMed2006

Proteomic analysis of antigens from Leishmania infantum promastigotes.

Leishmaniasis is a zoonotic disease caused by the species of the genus Leishmania, flagellated protozoa that multiply inside mammalian macrophages and are transmitted by the bite of the sandfly. The disease is widespread and due to the lack of fully effective treatment and vaccination the search for new drugs and immune targets is needed. Proteomics seems to be a suitable strategy because the annotated sequenced genome of L. major is available. Here, we present a high-resolution proteome for L. infantum promastigotes comprising of around 700 spots. Western blot with rabbit hyperimmune serum raised against L. infantum promastiogote extracts and further analysis by MALDI-TOF and MALDI-TOF/TOF MS allowed the identification of various relevant functional antigenic proteins. Major antigenic proteins were identified as propionil carboxilasa, ATPase beta subunit, transketolase, proteasome subunit, succinyl-diaminopimelate desuccinylase, a probable tubulin alpha chain, the full-size heat shock protein 70, and several proteins of unknown function. In addition, one enzyme from the ergosterol biosynthesis pathway (adrenodoxin reductase) and the structural paraflagellar rod protein 3 (PAR3) were found among non-antigenic proteins. This study corroborates the usefulness of proteomics in identifying new proteins with crucial biological functions in Leishmania parasites.

Animals↗
PubMed2006

Mass spectrometry-based proteomics for the detection of plant pathogens.

Plant diseases caused by fungi, oomycetes, viruses, and bacteria are devastating both to the economy and to the food supply of a nation. Therefore, the development of new, rapid methods to identify these pathogens is a highly important area of research that is of international concern. MS-based proteomics has become a powerful and increasingly popular approach to not only identify these pathogens, but also to better understand their biology. However, there is a distinction between identifying a pathogen protein and identifying a pathogen based upon the detection of one of its proteins and this must be considered before the general application of MS for plant pathogen detection is made. There has been a recent push in the proteomics community to make data from large-scale proteomics experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MS as a universal plant pathogen detection technology.

Bacterial Proteins↗