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PubMed2003

Rice proteomics: current status and future perspectives.

Rice, the first cereal crop genome to be decoded, has attracted the attention of researchers worldwide because of its immense socio-economic impact on human existence. With the availability of the draft genome sequence of two major types, japonica- and indica-rice, "rice proteomics" has entered into the era of functional genomics. Although during the last decade an important but limited progress (mainly construction of protein datafiles) has been made in the field of rice proteomics, it is only recently that dedicated research groups have taken this challenge to systematically analyze the rice proteome at the cell (and organelle), tissue, and whole plant level. Important gains achieved by the accelerated technological progress in protein separation and identification will help in going beyond the simple cataloguing of rice proteins in realistic terms. In this review, we discuss the progress made in the field of rice proteomics to date and dwell upon the future direction/problems/approaches towards defining the rice proteome.

Adaptation, Physiological↗

PubMed2004

Recent advances in capillary separations for proteomics.

The sequencing of several organisms' genomes, including the human's one, has opened the way for the so-called postgenomic era, which is now routinely coined as "proteomics". The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists of protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Still, the 2-D PAGE-mass spectrometry (MS) approach remains lacking in proteome coverage (for proteins having extreme isoelectric points or molecular masses as well as for membrane proteins), dynamic range, sensitivity, and throughput. Consequently, considerable efforts have been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings in 2-D PAGE while reserving the ability to resolve complex protein and peptide mixtures prior to MS analysis. This review focuses on the most recent advances in capillary-based separation techniques, including capillary liquid chromatography, capillary electrophoresis, and capillary electrokinetic chromatography, and combinations of multiples of these mechanisms, along with the coupling of these techniques to MS. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state-of-the-art integrated proteome effort. Miniaturized and integrated multidimensional peptide/protein separations using microfluidics are further summarized for their potential applications in high-throughput protein profiling toward biomarker discovery and clinical diagnosis.

Chromatography, Liquid↗

PubMed2006

Proteomics-driven progress in neurodegeneration research.

Proteomics technologies have been widely used in the investigation of neurodegenerative and psychiatric disorders, and in particular in the detection of differences between healthy individuals and patients suffering from such diseases. Thus, brain and cerebrospinal fluid (CSF) samples from patients with Alzheimer's disease, Down syndrome, Pick's disease, Parkinson's disease, schizophrenia, and other disorders as well as brain and CSF from animals serving as models of neurological disorders have been analyzed by proteomics. 2-DE followed by MALDI-TOF-MS has been mainly applied as this proteomics approach provides the possibility of convenient quantification of protein levels and detection of post-translational modifications. About 330 unique proteins with deranged levels and modifications have been detected by proteomics approaches to be related to neurodegeneration and psychiatric disorders. They are mainly involved in metabolism pathways, cytoskeleton formation, signal transduction, guidance, detoxification, transport, and conformational changes. In this article, we provide a summary of the major contributions of proteomics technologies in the study of neurodegenerative and psychiatric diseases, in particular, in the detection of changes in protein levels and modifications related to these disorders.

Animals↗

PubMed2006

Proteomic analysis of cartilage- and bone-associated samples.

The skeleton of the human body is built of cartilage and bone, which are tissues that contain extensive amounts of extracellular matrix (ECM). In bone, inorganic mineral hydroxyapatite forms 50-70% of the whole weight of the tissue. Although the organic matrix of bone consists of numerous proteins, 90% of it is composed of type I collagen. In cartilage, ECM forms a major fraction of the tissue, type II collagen and aggrecans being the most abundant macromolecules. It is obvious that the high content of ECM components causes analytical problems in the proteomic analysis of cartilage and bone, analogous to those in the analysis of low-abundance proteins present in serum. The massive contents of carbohydrates present in cartilage proteoglycans, and hydroxyapatite in bone, further complicate the situation. However, the development of proteomic tools makes them more and more tempting also for research of musculoskeletal tissues. Application of proteomic techniques to the research of chondrocytes, osteoblasts, osteocytes, and osteoclasts in cell cultures can immediately benefit from the present knowledge. Here we make an overview to previous proteomic research of cartilage- and bone-associated samples and evaluate the future prospects of applying proteomic techniques to investigate key events, such as cellular signal transduction, in cartilage- and bone-derived cells.

Amino Acid Sequence↗

PubMed2006

Divide and conquer: rat liver tissue proteomics based on the analysis of purified constituents.

Comparative proteome data of normal and diseased tissue samples are difficult to interpret. Proteins detected in tissues are derived from different cell types and blood constituents. Pathologic or toxicant-induced aberrations may affect the proteome profile of tissues in several ways since different cell types may respond in very different and highly specific manners. The aim of this study was to analyze the proteome profiles of purified rat liver primary cells and of blood plasma in comparison to liver whole tissue. Moreover, we investigated alterations of these profiles induced by the liver toxicant N-nitrosomorpholine (NNM) used as a model compound. Whole liver samples, pure hepatocytes and Kupffer cells as well as blood plasma were obtained from saline- or NNM-treated rats. Proteins were separated by 2-D PAGE and their amounts were estimated by fluorography. Selected proteins were identified by MS analysis of tryptic digests. Among them we identified proteins exclusively expressed in the analysed constituents. Several of these proteins were assigned in the proteome profile of whole-tissue homogenates. Furthermore, we identified several proteins that were modified, up-regulated or down-regulated due to NNM treatment in total liver homogenates. Some of these protein alterations were specifically detected in primary cells isolated from NNM-treated rats. Thus, we demonstrated the successful assignment of NNM-induced proteome alterations in rat liver to the cell type of origin. The currently applied approach may help to better understand pathologic processes at a whole-tissue level.

Animals↗

PubMed2006

Proteomic analysis of microglial contribution to mouse strain-dependent dopaminergic neurotoxicity.

Although the pathogenesis of Parkinson's disease (PD) remains unknown, it appears that microglial activation is associated with enhanced neurodegeneration in animal models of PD as well as in PD patients. Experimentally, C57BL/6 and SWR/J mice demonstrate striking differences in the extent of dopaminergic (DAergic) neurodegeneration induced by a parkinsonian toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The purpose of this study was to determine whether differences in microglial activation between these two strains of mice could provide insight into the variability seen in toxicant induced neuronal death, and subsequently to use a high-throughput proteomic method, combining stable isotope labeling with amino acids in cell culture (SILAC) with liquid chromatography and tandem mass spectrometry, to compare the microglial proteomes of C57BL/6 and SWR/J mice after stimulation with a classical microglial activator, lipopolysaccharide (LPS). We found that DAergic neurotoxicity induced by LPS in a primary neuron-microglia coculture was twofold greater with microglia isolated from the brains of C57BL/6 mice compared with that of SWR/J mice. Upon proteomic analysis we found that, out of over 1,000 proteins identified and quantified, 400 displayed a significant difference in their relative abundance between these two murine strains. Several proteins, which had relatively higher levels in C57BL/6 mice, have previously been implicated in LPS-mediated microglial activation, including those involved in the COX-2 pathway and in prostaglandin E-2 (PGE(2)) production. To validate our proteomic results we confirmed the increased expression level of iNOS in C57BL/6 vs. SWR/J microglia with semiquantitative Western blot. Further analysis of our proteomic discovery data will likely reveal numerous novel proteins involved in inflammation-mediated neurotoxicity in PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2006

Proteomics: clinical applications for head and neck squamous cell carcinoma.

The emerging field of proteomics offers great promise for unraveling the complex molecular events of tumorigenesis, as well as those that control clinically important tumor behaviors such as metastases, invasion, and resistance to therapy. Understanding the molecular basis of these tumor characteristics will usher in a new age of individualized cancer therapy. Advances in genomics, the study of genes and gene expression, as well as technologic advances have made possible study of all expressed proteins or the proteome (proteomics). Whereas genomics offers the opportunity to examine gene expression or variation in gene sequence, proteomics encompasses evaluation of protein expression, activation, modification, and degradation and ambitiously targets protein function. Although the present age has already been referred to as the post-genomic era, it is our opinion that both proteomics and genomics will provide clinically useful and complementary information that will speed scientific understanding of cancer. Ultimately, these new technologies will positively impact care of patients with head and neck squamous cell carcinoma.

Carcinoma, Squamous Cell↗

PubMed2006

HepatoProteomics: applying proteomic technologies to the study of liver function and disease.

The wealth of human genome sequence information now available, coupled with technological advances in robotics, nanotechnology, mass spectrometry, and information systems, has given rise to a method of scientific inquiry known as functional genomics. By using these technologies to survey gene expression and protein production on a near global scale, the goal of functional genomics is to assign biological function to genes with currently unknown roles in physiology. This approach carries particular appeal in disease research, where it can uncover the function of previously unknown genes and molecular pathways that are directly involved in disease progression. With this knowledge may come improved diagnostic techniques, prognostic capabilities, and novel therapeutic approaches. In this regard, the continuing evolution of proteomic technologies has resulted in an increasingly greater impact of proteome studies in many areas of research and hepatology is no exception. Our laboratory has been extremely active in this area, applying both genomic and proteomic technologies to the analysis of virus-host interactions in several systems, including the study of hepatitis C virus (HCV) infection and HCV-associated liver disease. Since proteomic technologies are foreign to many hepatologists (and to almost everyone else), this article will provide an overview of proteomic methods and technologies and describe how they are being used to study liver function and disease.

Humans↗

PubMed2002

Monoclonal and polyclonal humoral immune response to EC HER-2/NEU peptides with low similarity to the host's proteome.

We are studying peptide immunogenicity as a function of the similarity level to the host's proteome. By using as a model the breast/prostate cancer-associated HER-2/neu antigen, we analyzed the monoclonal and polyclonal humoral immune responses against HER-2/neu peptide motifs not shared with the host proteome. We show here that (i) a mouse monoclonal antibody (MAb) raised against the extracellular domain (EC) of human HER-2/neu oncoprotein recognized a linear peptide motif endowed with low similarity level to the mouse proteome; (ii) likewise, human sera from breast/prostate cancer patients preferentially recognized peptide fragments from the EC of the HER-2/neu oncoprotein having sequences that are not present in the human proteome. Together with previous results obtained in other disease models (cervical cancer-associated HPV16 E7 oncoprotein and Pemphigus vulgaris auto-antigen desmoglein-3), the present data suggest that a low level of sequence similarity to the host's proteome might be an important factor in shaping the pool of B cell epitopes.

Animals↗

PubMed2005

Rapid detection of fetal aneuploidy using proteomics approaches on amniotic fluid supernatant.

OBJECTIVE: Conventional chromosomal studies or fluorescent in situ hybridization takes days to diagnose fetal aneuploidies during amniocentesis. Here, we evaluated the value of mass spectrometry-based clinical proteomics analysis on amniotic fluid supernatant (AFS) as a rapid detection of fetal aneuploidies. METHODS: Proteomics profiles generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after fractionating samples with functionalized magnetic beads were used for differentiating 60 normal karyotypic from 20 aneuploid AFS. After the discriminating models were generated using genetic algorithm, we evaluated the clinical efficacy of the models in detecting aneuploidies in two batches (each n=30) of AFS prior to the release of chromosomal diagnoses. RESULTS: Within hours, the two-step proteomics analysis of AFS with the C18 model, followed by the weak cation exchange model, was able to detect aneuploid AFS at 3.3% disease prevalence rate with 100% sensitivity, 72 to 96% specificity, 11 to 50% positive predictive value, and 100% negative predictive value. CONCLUSION: Clinical proteomics analysis of AFS using magnetic beads-based sample preparation and MALDI-TOF-MS can be used as a rapid detection for fetal aneuploidies. With perfect sensitivity and negative predictive value of the two-step proteomics method, it may be used for rapid detection of aneuploid AFS immediately after amniocentesis. Further large-scale examinations are apparently needed to verify the clinical value of this rapid detection.

Amniotic Fluid↗

PubMed2003

The proteomics standards initiative.

The Proteomics Standards Initiative (PSI) aims to define community standards for data representation in proteomics and to facilitate data comparison, exchange and verification. Progress has been made in the development of common standards for data exchange in the fields of both mass spectrometry and protein-protein interaction. A proteomics-specific extension is being created for the emerging American Society for Tests and Measurements mass spectrometry standard, which will be supported by manufacturers of both hardware and software. A data model for proteomics experimentation is under development and discussions on a public repository for published proteomics data are underway. The Protein-Protein Interactions group expects to publish the Level 1 PSI data exchange format for protein-protein interactions soon and discussions as to the content of Level 2 have been initiated.

Biochemistry↗

PubMed2004

Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry.

Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.

Blood Platelets↗

PubMed2004

ProteomeGRID: towards a high-throughput proteomics pipeline through opportunistic cluster image computing for two-dimensional gel electrophoresis.

The quest for high-throughput proteomics has revealed a number of critical issues. Whilst improved two-dimensional gel electrophoresis (2-DE) sample preparation, staining and imaging issues are being actively pursued by industry, reliable high-throughput spot matching and quantification remains a significant bottleneck in the bioinformatics pipeline, thus restricting the flow of data to mass spectrometry through robotic spot excision and protein digestion. To this end, it is important to establish a full multi-site Grid infrastructure for the processing, archival, standardisation and retrieval of proteomic data and metadata. Particular emphasis needs to be placed on large-scale image mining and statistical cross-validation for reliable, fully automated differential expression analysis, and the development of a statistical 2-DE object model and ontology that underpins the emerging HUPO PSI GPS (Human Proteome Organization Proteomics Standards Initiative General Proteomics Standards). The first step towards this goal is to overcome the computational and communications burden entailed by the image analysis of 2-DE gels with Grid enabled cluster computing. This paper presents the proTurbo framework as part of the ProteomeGRID, which utilises Condor cluster management combined with CORBA communications and JPEG-LS lossless image compression for task farming. A novel probabilistic eager scheduler has been developed to minimise make-span, where tasks are duplicated in response to the likelihood of the Condor machines' owners evicting them. A 60 gel experiment was pair-wise image registered (3540 tasks) on a 40 machine Linux cluster. Real-world performance and network overhead was gauged, and Poisson distributed worker evictions were simulated. Our results show a 4:1 lossless and 9:1 near lossless image compression ratio and so network overhead did not affect other users. With 40 workers a 32x speed-up was seen (80% resource efficiency), and the eager scheduler reduced the impact of evictions by 58%.

Algorithms↗

PubMed2003

Progress in the definition of a reference human mitochondrial proteome.

Owing to the complexity of higher eukaryotic cells, a complete proteome is likely to be very difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and "physiology" of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting. The optimization steps in two-dimensional electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies.

Databases as Topic↗

PubMed2004

A comprehensive proteome map of growing Bacillus subtilis cells.

The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for its application in microbial physiology. DNA arrays were used to calculate the number of genes transcribed in growing cells. From the 4100 B. subtilis genes, 2515 were actively transcribed in cells grown under standard conditions. From these genes 1544 proteins should be covered by our standard gel system pI 4-7. Using this standard gel system and supplementary zoom gels (pI 5.5-6.7, 5-6, 4.5-5.5, and 4-5) 693 proteins which are expressed in growing cells were detected that cover more than 40% of the vegetative proteome predicted for this region. Particularly broad coverage and thus comprehensive monitoring will be possible for central carbohydrate metabolism (glycolysis, pentose phosphate shunt, and citric acid cycle), amino acid synthesis pathways, purine and pyrimidine metabolism, fatty acid metabolism, and main cellular functions like replication, transcription, translation, and cell wall synthesis. Comparing the theoretical pI and Mr values with those experimentally determined a reasonable correlation was found for the majority of protein spots. By a color code outliers with dramatic deviations in charge or mass were visualized that may indicate post-translational modifications. In addition to the cytosolic neutral and alkaline proteins, 130 membrane proteins were found relying on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) techniques. The vegetative proteome containing 876 proteins in total is now ready for physiological applications. Two main proteome fractions (pI 4-7 and zoom gel pI 4.5-5.5) should be sufficient for such high-throughput physiological proteomics.

Bacillus subtilis↗

PubMed2005

Rice proteome analysis: a step toward functional analysis of the rice genome.

The technique of proteome analysis using 2-DE has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this review, we describe construction of the rice proteome database, the cataloging of rice proteins, and the functional characterization of some of the proteins identified. Initially, proteins extracted from various tissues and organelles were separated by 2-DE and an image analyzer was used to construct a display or reference map of the proteins. The rice proteome database currently contains 23 reference maps based on 2-DE of proteins from different rice tissues and subcellular compartments. These reference maps comprise 13 129 rice proteins, and the amino acid sequences of 5092 of these proteins are entered in the database. Major proteins involved in growth or stress responses have been identified by using a proteomics approach and some of these proteins have unique functions. Furthermore, initial work has also begun on analyzing the phosphoproteome and protein-protein interactions in rice. The information obtained from the rice proteome database will aid in the molecular cloning of rice genes and in predicting the function of unknown proteins.

Calreticulin↗

PubMed2005

Perspectives in spicing up proteomics with splicing.

In the post-genomics era there has been an acceleration of understanding of cellular and organismal biology and this acceleration has moved the goalposts for proteomics. Higher eukaryotes use alternative promoters, alternative splicing, RNA editing and post-translational modification to produce multiple isoforms of proteins from single genes. Switching amongst these isoforms is a major mechanism for control of cellular function. At present fundamental limitations in sensitivity, in absolute quantitation of proteins and in the characterization of protein structure at functionally important levels strongly limit the applicability of proteomics to higher eukaryotes. Recent developments suggest that quantitative, top-down proteomics analyses of complete proteins at sub-attomole levels are necessary for physiologically relevant studies of higher eukaryotes. New proteomics technologies which will ensure the future of proteomics as an important technology in medicine and cellular biology of higher eukaryotes are becoming available.

Alternative Splicing↗

PubMed2005

Distinct proteome features of plasma microparticles.

Plasma microparticles (MPs) are spherical cell membrane fragments derived from either apoptotic or activated cells. Characterized by a rich phospholipid moiety and many protein constituents, MPs normally circulate in the blood and contribute to numerous physiological processes. In disease states, MPs derived from the injured organ likely contain valuable markers for determining the site, type, and extent of disease pathology. However, the basic protein characteristics of plasma MPs have yet to be described. In this study, MPs from a pooled plasma sample derived from 16 healthy donors, all of group A blood type, were prepared by ultracentrifugation. Flow cytometry confirmed that a majority of these MPs are smaller than 1 microm. Factor Xa generation assay revealed the presence of tissue factor activity in these MPs, confirming MPs' role in initiating blood coagulation. The MP proteome was analyzed by two-dimensional (2-D) gel electrophoresis performed in triplicate, and compared with a 2-D gel of pooled whole plasma and blood platelets. Overall, plasma MPs displayed distinct protein features and a greater number of protein spots (1021-1055) than that detected in whole plasma (331-370). Protein spots expressed in high abundance in the MP proteome were then excised and submitted for protein identity determination. This process provided protein identification for 169 protein spots and reported their relative protein quantities within the MP proteome. These 169 protein spots represented 83 different proteins and their respective isoforms. Thirty of these proteins have never before been reported in previous proteome analyses of human plasma. These results provide unprecedented information on the MP proteome and create a basis for future studies to understand MP biology and pathophysiology.

Adolescent↗