[Show abstract][Hide abstract] ABSTRACT: Proteomic technologies, such as yeast twohybrid, mass spectrometry (MS), protein/ peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories. Even more difficult is the annotation and display of pertinent information in the context of the corresponding proteins. Wikipedia, an online encyclopedia that anyone can edit, has already proven quite successful1 and can be used as a model for sharing biological data. However, the need for experimental evidence, data standardization and ownership of data creates scientific obstacles.
[Show abstract][Hide abstract] ABSTRACT: Proteomics-based studies offer a powerful complementary approach to DNA/RNA-based investigations and are now being applied to investigate aspects of many diseases including cancer. However, the heterogeneous nature of tissue samples often makes interpretation difficult. We have undertaken a study into the potential use of a novel laser capture microdissection (LCM) system to isolate cells of interest for subsequent proteomic analysis. Retrieval of selected cells is achieved by activation of a transfer film placed in contact with a tissue section, by a laser beam (30 or 60 microm diameter) which is focused on a selected area of tissue using an inverted microscope. The precise area of film targeted by the laser bonds to the tissue beneath it and these cells are then lifted free of surrounding tissue. Although the technique has been shown to be readily compatible with subsequent analysis of nucleic acids, little information is yet available regarding the application of protein-based analyses to the captured tissue. We report here preliminary data regarding the potential use of the LCM system in combination with two-dimensional electrophoresis to examine protein profiles of selected tissue areas. Electrophoretic profiles of proteins from normal and malignant renal tissue samples showed little change following LCM, nine selected proteins showed identical mass spectrometric sequencing profiles, and two selected proteins retained antigenicity. Dissection of epithelial tissue from a sample of normal human cervix resulted in enrichment of some proteins compared with analysis of the whole tissue. LCM will be a valuable adjunct to proteomic studies although further detailed validation is necessary.
[Show abstract][Hide abstract] ABSTRACT: There is evidence for both similarity and distinction in the presentation and molecular characterization of schizophrenia and bipolar disorder. In this study, we characterized protein abnormalities in the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder using two-dimensional gel electrophoresis. Tissue samples were obtained from 35 individuals with schizophrenia, 35 with bipolar disorder and 35 controls. Eleven protein spots in schizophrenia and 48 in bipolar disorder were found to be differentially expressed (P<0.01) in comparison to controls, with 7 additional spots found to be altered in both diseases. Using mass spectrometry, 15 schizophrenia-associated proteins and 51 bipolar disorder-associated proteins were identified. The functional groups most affected included synaptic proteins (7 of the 15) in schizophrenia and metabolic or mitochondrial-associated proteins (25 of the 51) in bipolar disorder. Six of seven synaptic-associated proteins abnormally expressed in bipolar disorder were isoforms of the septin family, while two septin protein spots were also significantly differentially expressed in schizophrenia. This finding represented the largest number of abnormalities from one protein family. All septin protein spots were upregulated in disease in comparison to controls. This study provides further characterization of the synaptic pathology present in schizophrenia and of the metabolic dysfunction observed in bipolar disorder. In addition, our study has provided strong evidence implicating the septin protein family of proteins in psychiatric disorders for the first time.
[Show abstract][Hide abstract] ABSTRACT: Skeletal muscle fibre transitions occur in many biological processes, in response to alterations in neuromuscular activity, in muscular disorders, during age-induced muscle wasting and in myogenesis. It was therefore of interest to perform a comprehensive proteomic profiling of muscle transformation. Chronic low-frequency stimulation of the rabbit tibialis anterior muscle represents an established model system for studying the response of fast fibres to enhanced neuromuscular activity under conditions of maximum activation. We have conducted a DIGE analysis of unstimulated control specimens versus 14- and 60-day conditioned muscles. A differential expression pattern was observed for 41 protein species with 29 increased and 12 decreased muscle proteins. Identified classes of proteins that are changed during the fast-to-slow transition process belong to the contractile machinery, ion homeostasis, excitation-contraction coupling, capillarization, metabolism and stress response. Results from immunoblotting agreed with the conversion of the metabolic, regulatory and contractile molecular apparatus to support muscle fibres with slower twitch characteristics. Besides confirming established muscle elements as reliable transition markers, this proteomics-based study has established the actin-binding protein cofilin-2 and the endothelial marker transgelin as novel biomarkers for evaluating muscle transformation.
[Show abstract][Hide abstract] ABSTRACT: Both the generation and the analysis of proteomics data are now widespread, and high-throughput approaches are commonplace. Protocols continue to increase in complexity as methods and technologies evolve and diversify. To encourage the standardized collection, integration, storage and dissemination of proteomics data, the Human Proteome Organization's Proteomics Standards Initiative develops guidance modules for reporting the use of techniques such as gel electrophoresis and mass spectrometry. This paper describes the processes and principles underpinning the development of these modules; discusses the ramifications for various interest groups such as experimentalists, funders, publishers and the private sector; addresses the issue of overlap with other reporting guidelines; and highlights the criticality of appropriate tools and resources in enabling 'MIAPE-compliant' reporting.
[Show abstract][Hide abstract] ABSTRACT: Pulmonary fibrosis arises as a consequence of aberrant remodeling and defective repair mechanisms within the lung. This destructive process is the cause of much of the morbidity and mortality in many pulmonary disorders. Unfortunately, therapeutic options are limited. A significant advancement in the management of patients with pulmonary fibrosis would be the identification of biomarkers for diagnosis, prognosis and prediction of patient response to therapy. Bronchoalveolar lavage is an ideal tissue target for the discovery of these potential biomarkers in pulmonary fibrosis. Integrative approaches using both gel- and mass spectrometry-based proteomic workflows will allow full coverage of this complex proteome, thereby unlocking this potential information as a clinical tool to aid diagnosis and guide treatment for individual patients with pulmonary fibrosis.
Expert Review of Proteomics 07/2007; 4(3):379-88. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In a proteomic approach using 2-DE, the changes in protein expression patterns in wing imaginal discs induced by hormone treatment have been studied. Here we show the response of butterfly imaginal wing disc tissue taken from late fifth instar larvae of the African-Mocker swallowtail Papilio dardanus (Lepidoptera) to the insect hormones 20-hydroxyecdysone (20-HE) and juvenile hormone (JH). The tissues were cultured in the presence of one hormone or a combination of both and their protein expression was compared to the pattern obtained from untreated wing discs. All the treatments resulted in changes in the expression pattern distinct from the uninduced control, indicating a distinct protein regulation induced by the hormones. The treatment with both of the hormones, which are known to have antagonistic physiological effects, did show a unique pattern, presumably the result, in part, of synergistic effects on protein expression mediated by the combined effects of both the hormones. The extent of the interaction between JH and 20-HE indicates a complex molecular regulation, far beyond a simple antagonistic effect.
[Show abstract][Hide abstract] ABSTRACT: Prostate cancer is the commonest solid-organ malignancy to affect men in Europe and the USA; it is estimated that one in six men will develop this cancer in their lifetime. Current screening relies on a digital rectal examination with a serum prostate-specific antigen test. Novel urinary diagnostic tests are potentially interesting screening tools for this disease. We examined published reports assessing the use of urinary markers for the diagnosis of prostate cancer. Using a PubMed-based search we identified studies of urinary markers for prostate cancer published from 1985 to February 2006 using the search terms 'urine', 'marker' and 'prostate cancer'. Studies to date have used small cohorts and relied on prostatic biopsies to provide histology. The sensitivity and specificity of markers are wide ranging but with only a few studies published on each putative marker it is difficult to assess their potential impact. Using urinary biomarkers for prostate cancer is a relatively novel diagnostic approach; they are appealing as a screening test because they are not invasive. Further work is needed to identify and validate 'signature markers' indicative of prostatic malignancy. The newer proteomic platforms are promising biomarker discovery tools that might uncover the next generation of urinary biomarkers.
BJU International 03/2007; 99(2):263-8. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The majority of cardiovascular proteomic investigations reported to date have employed two-dimensional gel electrophoresis (2DE) with immobilized pH gradients to separate the sample proteins, combined with quantitative computer analysis to detect differentially expressed proteins and mass spectrometry technologies to identify proteins of interest. In spite of the development of novel gel-free technologies, 2DE remains the only technique that routinely can be applied to parallel quantitative expression profiling of large sets of complex protein mixtures, such as those represented by cardiovascular cell and tissue lysates. This chapter details a procedure for large-format 2DE, and its variations, that has been successfully applied in cardiovascular proteomic research.
Methods in Molecular Biology 02/2007; 357:3-13. · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of ECL-Plex CyDye-conjugated secondary antibodies allows the advancement of conventional Western blotting, opening up possibilities for highly sensitive and quantitative protein confirmation and identification. We report a novel proteomic method to simultaneously visualise the total protein profile as well as the specific immunodetection of an individual protein species by combining cyanine CyDye pre-labelled proteins and antibody immunoblotting. This technique proposes to revolutionise both 2-D immunoprobing and protein confirmation following MS analysis.
[Show abstract][Hide abstract] ABSTRACT: The Human Proteome Organisation (HUPO) Brain Proteome Project (BPP) pilot studies have generated over 200 2-D gels from eight participating laboratories. This data includes 67 single-channel and 60 DIGE gels comparing 30 whole frozen C57/BL6 female mouse brains, ten each at embryonic day 16, postnatal day 7 (juvenile) and postnatal day 54-56 (adult); and ten single-channel and three DIGE gels comparing human epilepsy surgery of the temporal front lobe with a corresponding post-mortem specimen. The samples were generated centrally and distributed to the participating laboratories, but otherwise no restrictions were placed on sample preparation, running and staining protocols, nor on the 2-D gel analysis packages used. Spots were characterised by MS and the annotated gel images published on a ProteinScape web server. In order to examine the resultant differential expression and protein identifications, we have reprocessed a large subset of the gels using the newly developed RAIN (Robust Automated Image Normalisation) 2-D gel matching algorithm. Traditional approaches use symbolic representation of spots at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in spot modelling and matching. With RAIN, image intensity distributions, rather than selected features, are used, where smooth geometric deformation and expression bias are modelled using multi-resolution image registration and bias-field correction. The method includes a new approach of volume-invariant warping which ensures the volume of protein expression under transformation is preserved. An image-based statistical expression analysis phase is then proposed, where small insignificant expression changes over one gel pair can be revealed when reinforced by the same consistent changes in others. Results of the proposed method as applied to the HUPO BPP data show significant intra-laboratory improvements in matching accuracy over a previous state-of-the-art technique, Multi-resolution Image Registration (MIR), and the commercial Progenesis PG240 package.
[Show abstract][Hide abstract] ABSTRACT: Although the number of patients receiving successful transplantation as a means of treatment for end-stage organ failure continues to increase, rejection, in particular chronic rejection still poses a major risk factor. Crucially, only 50% of allografted hearts and kidneys survive for 10 years. Currently, biopsy is the most common and, in some instances, the only means of diagnosing rejection: it is both invasive and costly. A blood or urine biomarker would overcome these problems and conceivably enable diagnosis of rejection far earlier than a biopsy because it could be diagnosed before clinical symptoms. Furthermore, development of a biomarker could lead to new insights into the disease process. In recent years, proteomics has provided a novel means of examining potential biomarkers. We discuss here the various proteomic platforms available, including 2-dimensional gel electrophoresis, mass spectrometry, and protein arrays, and the various studies exploring protein profiling in heart, lung, kidney, and liver transplantation.
[Show abstract][Hide abstract] ABSTRACT: The Human Proteome Organisation (HUPO) initiated several projects focusing on the proteome analysis of distinct human organs. The Brain Proteome Project (BPP) is the initiative dedicated to the brain, its development and correlated diseases. Two pilot studies have been performed aiming at the comparison of techniques, laboratories and approaches. With the help of the results gained, objective data submission, storage and reprocessing workflow have been established. The biological relevance of the data will be drawn from the inter-laboratory comparisons as well as from the re-calculation of all data sets submitted by the different groups. In the following, results of the single groups as well as the centralised reprocessing effort will be summarised and compared, showing the added value of this concerted work.
[Show abstract][Hide abstract] ABSTRACT: Brain development and aging is a complex process involving proliferation, differentiation and apoptosis. Elucidating proteome changes in these processes can help to understand the mechanisms of brain development and maintenance as well as neurodegenerative diseases. The research reported here is a contribution to the HUPO Brain Proteome Project mouse pilot study. Whole, frozen C57BL/6J mouse brain comprising three different developmental stages (embryonic day 16, postnatal day 7, and postnatal days 54-58) were processed by using 2-D DIGE. A total of 1999 spots were matched between all gels. Of these, 206 spots were differentially expressed between the different stages: 122 spots were highest in intensity in embryonic stage E16, 26 highest in the juvenile group P7 and 58 spots highest in P56, the adult stage. The results show a pattern of temporal expression. Based on the expression patterns we tentatively suggest that proteins involved in the establishment of primary structures in the brain are expressed highest in the embryonic mouse. Proteins involved in the development of the brain are expressed highest in the juvenile phase and proteins that make utilization of the brain possible by delivering energy are expressed highest in the adult mice.
[Show abstract][Hide abstract] ABSTRACT: The p21Waf1/Cip1/Sdi1 cyclin-dependent kinase inhibitor is a key regulator of cell cycle progression and has also been observed to influence the expression of genes associated with several age-related disorders. Previous work has shown that expression of p21 in tumour cells mediates an antiapoptotic and mitogenic paracrine effect, which is in contrast to the arrested state of p21-expressing cells. Here, we have employed SELDI-MS technology to characterise, at a proteomic level, factors released from HT-1080 human fibrosarcoma cells displaying inducible p21 expression. Conditioned media from induced and noninduced cells were profiled on a range of diverse ProteinChip arrays and subjected to SELDI-MS analysis. Evaluation of proteins binding onto IMAC, Q10 or CM10 surfaces led to the discovery of a number of putative p21-regulated factors. We further validated three p21-regulated proteins observed at 10.2, 11.7 and 13.4 kDa. Using Q Ceramic HyperD fractionation columns, we were able to selectively enrich for each of these three proteins. Subsequent SDS-PAGE and MS analysis of tryptic digests identified the 13.4 kDa protein as cystatin C and the 10.2 kDa protein as pro-platelet basic protein (PPBP). Judging by the apparent MW and the pI of the 11.7 kDa protein, we reasoned that it may be beta-2-microglobulin, which was confirmed by subsequent identification. Increased levels of cystatin C and beta-2-microglobulin in conditioned media from p21-expressing cells was confirmed by antibody capture experiments using anticystatin C and anti-beta-2-microglobulin antibodies on preactivated PS-20 arrays. Western blot analysis demonstrated increased expression of intracellular and extracellular cystatin C and beta-2-microglobulin in p21-expressing cells, compared to noninduced controls. Increased levels of PPBP were validated in cell lysates from p21-expressing cells. The three secreted factors that we have identified in this study, have all been shown previously to have growth modulating effects and, as such, may contribute to the observed mitogenic and anti-apoptotic paracrine activity of p21-expressing [corrected] cells.
[Show abstract][Hide abstract] ABSTRACT: More than 70 interested colleagues attended the 5th Workshop of the HUPO Brain Proteome Project (HUPO BPP) at the UCD Conway Institute, Dublin, Ireland. An overview of the outcome of the pilot study was presented and the new subprojects "Clinical Neuroproteomics of Human Body Fluids" as well as "Cerebellum 2D-mapping" were announced. In addition the election of the HUPO BPP committees and the future directions of this project were discussed and decided. The meeting was enhanced by several talks highlighting the application of proteomics in biomedical research.
[Show abstract][Hide abstract] ABSTRACT: Biomarkers for neurodegenerative disorders are potentially present in cerebrospinal fluid (CSF) and can be detected using proteomic technologies. Since CSF is high in salt and low in protein, its study by proteomic methods requires appropriate sample preparation. In this study, we applied four different sample treatments to the same ovine CSF sample. Precipitation with acetone or using a 2-D Clean-Up Kit (GE Healthcare BioSciences, Little Chalfont, UK) preserved more proteins, and produced more gel spots than spin columns from Sigma and Bio-Rad. A 53-kDa spot, identified by MS/MS as transthyretin (TTR) tetramer, was not detected in samples treated with the 2-D Clean-Up Kit, though it was always present on all gels prepared using the other three methods. Western immunoblotting confirmed the low recovery of tetrameric TTR by the 2-D Clean-Up Kit and showed that the tetrameric form of TTR predominated in ovine but not in rat CSF. In one ovine CSF sample haemoglobin was found, indicating blood contamination. We conclude that acetone precipitation is a simple and efficient way to prepare ovine CSF for 2-DE. The use of the 2-D Clean-Up Kit leads to the disappearance of tetrameric TTR only from ovine CSF proteome.