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Results for “proteome mapping”

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PubMed2006

Growth-induced changes in the proteome of Helicobacter pylori.

Helicobacter pylori is a major human pathogen that is responsible for a number of gastrointestinal infections. We have used 2-DE to characterise protein synthesis in bacteria grown either on solid agar-based media or in each of two broth culture media (Brucella and brain heart infusion (BHI) broth). Significant differences were observed in the proteomes of bacteria grown either on agar-based or in broth media. Major changes in protein abundance were identified using principal component analysis (PCA), which delineated the profiles derived for the three key growth conditions (i.e. agar plates, Brucella and BHI broth). Proteins detected across the gel series were identified by peptide mass mapping and Edman sequencing. A number of proteins associated with protein synthesis in general as well as specific amino acid synthesis were depressed in broth-grown bacteria compared to plate-grown bacteria. A similar reduction was also observed in the abundance of proteins involved in detoxification. Two of the most abundant spots, identified as UreB and GroEL, in plate-grown bacteria showed a >140-fold drop in abundance in bacteria grown in Brucella broth compared to bacteria grown on agar plates. Two protein spots induced in bacteria grown in broth culture were both identified as glyceraldehyde 3-phosphate dehydrogenase based on their N-terminal amino acid sequences derived by Edman degradation. The underlying causes of the changes in the proteins abundance were not clear, but it was likely that a significant proportion of the changes were due to the alkaline pH of the broth culture media.

Agar↗
PubMed2005

Peeling the yeast protein network.

A set of highly connected proteins (or hubs) plays an important role for the integrity of the protein interaction network of Saccharomyces cerevisae by connecting the network's intrinsic modules. The importance of the hubs' central placement is further confirmed by their propensity to be lethal. However, although highly emphasized, little is known about the topological coherence among the hubs. Applying a core decomposition method which allows us to identify the inherent layer structure of the protein interaction network, we find that the probability of nodes both being essential and evolutionary conserved successively increases toward the innermost cores. While connectivity alone is often not a sufficient criterion to assess a protein's functional, evolutionary and topological relevance, we classify nodes as globally and locally central depending on their appearance in the inner or outer cores. The observation that globally central proteins participate in a substantial number of protein complexes which display an elevated degree of evolutionary conservation allows us to hypothesize that globally central proteins serve as the evolutionary backbone of the proteome. Even though protein interaction data are extensively flawed, we find that our results are very robust against inaccurately determined protein interactions.

Databases, Protein↗
PubMed2006

Proteomic responses of rice young panicles to salinity.

Rice (Oryza sativa) is most sensitive to salinity during the reproductive stage. We employed a proteomic approach to further understand the mechanism of plant responses to salinity at an early reproductive stage. Plants were grown in culture solution and salt stress imposed at panicle initiation. After 12 days of stress, young panicles were collected from control and salt stressed plants. The Na+ and K+ content of panicle and several yield components changed significantly in response to short-term salt stress. The collected panicles were sorted into three different sizes (7 +/- 1, 11 +/- 1, and 15 +/- 1 mm) and their proteome patterns were analyzed using 2-DE in triplicates. The expression pattern of 13 proteins significantly changed in all panicle sizes in response to stress. MS analysis of salt responsive proteins and 16 other highly abundant proteins of panicle led to the identification of proteins involved in several salt responsive mechanisms which may increase plant adaptation to salt stress including higher constitutive expression level and up-regulation of antioxidants, up-regulation of proteins involved in translation, transcription, signal transduction, and ATP generation. To the best of our knowledge, this is the first proteome analysis of plant young panicle which may enhance our understanding of plant molecular responses to salinity. Proteome reference map of rice young panicle is available at http://www.proteome.ir.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Proteome analysis of signaling cascades in human platelets.

During the last 15 years, advances in mass spectrometry (MS) instrumentation and techniques have revolutionized the emerging field of proteomics. Proteomics technology allows a comprehensive and efficient analysis of the protein content (i.e., the proteome) of any cell, tissue or biological fluid and has become an indispensable tool in biomedical research, complementing the powerful field of genomics. Proteomics is based on the huge analytical power offered by mass spectrometry in combination with several separation techniques, such as two-dimensional gel electrophoresis (2-DE) or multidimensional liquid chromatography. The technology is particularly suitable for platelets because of the absence of a nucleus. In the recent years, there has been success in mapping the proteome of the platelet in a basal state. Furthermore, a handful of research groups have also applied this technology to the study of signaling cascades in human platelets, allowing the identification of novel platelet signaling proteins and phosphorylation events. Those studies provide new insights into the mechanisms of platelet activation and build the basis for the development of therapeutic agents for thrombotic disease. This article focus on the application of 2-DE-based proteomics to the study of signaling cascades in human platelets.

Blood Platelets↗
PubMed2006

Technologies for bacterial surface proteomics.

Proteins from bacterial membranes are notoriously difficult to analyze using the traditional technologies encompassed under the term 'proteomics'. This is because of several factors, including the comparatively low abundance of most membrane proteins within a complex mixture containing cytoplasmic metabolic enzymes, the poor solubility of membrane components such as phospholipids, lipopolysaccharides and peptidoglycans, and the inherent hydrophobicity of many integral membrane proteins that contain up to 15 transmembrane-spanning regions. Recent advances in gel-based and chromatographic separations, coupled with protein and peptide labelling and the exquisite sensitivity of mass spectrometry, are finally beginning to overcome these problems. New technologies in membrane proteomics enable comparative analysis of these recalcitrant proteins from bacteria under a variety of biological conditions.

Bacteria↗
PubMed2003

Proteome analysis of diseased joints from mice suffering from collagen-induced arthritis.

Strains of mice that are susceptible to autoimmunity have provided experimental models to analyze the molecular basis for the complex multifactorial inheritance of human autoimmune disease. In this study proteins associated with collagen-induced arthritis (CIA) in mice were experimentally identified using a global proteomics approach. Two-dimensional gels of proteins from inflamed and non-inflamed joints showed a distinguished protein profile visualizing about 530 Coomassie-stained protein spots in the pH 4-7 range. A total of 76 spots were identified by peptide mass fingerprinting with good confidence. They included proteins of cytoskeletal origin, chaperones, enzymes and also some signal transduction molecules. Comparison to gels from non-inflamed paws pointed to proteins that were differentially expressed between the control and diseased state. Ferritin light chain and antioxidant protein 2 were slightly more abundant, lymphoid enhancer binding factor 1 slightly, but significantly, less abundant in inflamed paws. Fourteen of the identified proteins were the products of genes that had increased transcript levels in the diseased state. However, on the protein level no significant differences were found in comparison to the controls. This study provides us with the framework for more detailed approaches to understanding the complex disease arthritis that go beyond global proteomics.

Amino Acid Sequence↗
PubMed2000

A proteomic approach for the study of Saccharomyces cerevisiae cell wall biogenesis.

In fungi, cell shape is determined by the presence of a rigid cell wall which separates the cell from the extracellular medium. This highly dynamic structure is essential for the maintenance of cell integrity and is involved in several phenomena such as flocculation, adherence and pathogenicity. The composition of the fungal cell wall is well known, but issues such as the assembly and remodeling of its components remain poorly understood. In an attempt to study the de novo construction of the yeast cell wall, we have undertaken a large-scale proteomic approach to analyze the proteins secreted by regenerating protoplasts. Upon incubation of protoplasts in regenerating conditions, numerous proteins are secreted into the culture medium. These presumably include proteins destined for the cell wall, comprising both structural proteins as well as enzymes involved in cell wall biogenesis. This work reports the establishment of a reference map of proteins secreted by regenerating protoplasts by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and their identification by mass spectrometry. Thirty-two different proteins have been identified, including known cell wall proteins, glycolytic enzymes, heat shock proteins, and proteins involved in several other processes. Using this approach, novel proteins possibly involved in cell wall construction have also been identified. This reference map will allow comparative analyses to be carried out on a selected collection of mutants affected in the cell wall.

Amino Acid Sequence↗
PubMed2000

Quantitative proteome analysis: methods and applications.

With the completion of a rapidly increasing number of complete genomic sequences, much attention is currently focused on how the information contained in sequence databases might be interpreted in terms of the structure, function, and control of biological systems. Quantitative proteome analysis, the global analysis of protein expression, has been proposed as a method to study steady-state gene expression and perturbation-induced changes. Here, we discuss the rationale for quantitative proteome analysis, highlight the limitations in the current standard technology, and introduce a new experimental approach to quantitative proteome analysis.

Amino Acid Sequence↗
PubMed2006

Proteomic exploration of the Wnt/beta-catenin pathway.

The Wnt/beta-catenin pathway, with its substantial role in development and disease processes, has been the subject of intensive studies for more than two decades. The multiplicity of protein components in the pathway, with their complicated interactions, phosphorylations, and subcellular localization events, presents itself as a great system for proteomic analysis. Recent advances in proteomic technologies have enabled protein identification and quantification in complex protein mixtures at a global scale, leading to new insights into the pathway. This review will discuss several of the new proteomic technologies, and how they have been applied to the interrogation of the Wnt/beta-catenin pathway, with particular reference to the application of yeast two-hybrid analysis and mass spectrometry-based approaches.

Animals↗
PubMed2004

Proteomic analysis of rat atrial secretory granules: a platform for testable hypotheses.

The recent development of powerful proteomic tools has enabled investigators to directly examine the population of proteins present in defined biological systems. We report here the first proteomic analysis of atrial secretory granules. Approximately 100 distinct protein components of the atrial secretory granule proteome were detected using subcellular fractionation and one-dimensional SDS-PAGE in conjunction with peptide mass fingerprinting by MALDI-TOF mass spectrometry. Of this number, 61 proteins were clearly identified by high probability data matches and repeated observation. The majority of the proteome was found to be membrane-associated with the most prominent proteins being peptidylglycine alpha-amidating monooxygenase (PAM) and pro-atrial natriuretic peptide (pro-ANP). This proteomic analysis of the rat atrium secretory granule produced an assembly of proteins with a diverse array of reported functions. The identified proteins fall into seven functional categories: (1) granular transport, docking and fusion; (2) signal transduction; (3) calcium-binding/calcium-dependent; (4) cellular architecture/chaperoning; (5) peptide/protein processing; (6) hormone; (7) proton transport. The novel finding of several protein processing enzymes and signal transduction proteins offer new perspectives on how pro-ANP is stored and processed to ANP during release. Accordingly, defining the proteome of the atrial secretory granule provides a framework for the development of new hypotheses that address key mechanisms governing granule function and ANP secretion.

Animals↗
PubMed2000

Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology.

Proteome analysis is most commonly accomplished by a combination of two-dimensional gel electrophoresis (2DE) to separate and visualize proteins and mass spectrometry (MS) for protein identification. Although this technique is powerful, mature, and sensitive, questions remain concerning its ability to characterize all of the elements of a proteome. In the current study, more than 1,500 features were visualized by silver staining a narrow pH range (4.9-5. 7) 2D gel in which 0.5 mg of total soluble yeast protein was separated. Fifty spots migrating to a region of 4 cm(2) were subjected to MS protein identification. Despite the high sample load and extended electrophoretic separation, proteins from genes with codon bias values of <0.1 (lower abundance proteins) were not found, even though fully one-half of all yeast genes fall into that range. Proteins from genes with codon bias values of <0.1 were found, however, if protein amounts exceeding the capacity of 2DE were fractionated and analyzed. We conclude that the large range of protein expression levels limits the ability of the 2DE-MS approach to analyze proteins of medium to low abundance, and thus the potential of this technique for proteome analysis is likewise limited.

Amino Acid Sequence↗
PubMed2006

Proteomic analysis of fibrillin-rich microfibrils.

MS has been used to investigate the composition of fibrillin-rich microfibrils from non-elastic and elastic tissues, and to compare fibrillin-1 tryptic fingerprints derived from whole zonules, microfibrils and recombinant fibrillin-1. In all microfibril preparations, fibrillin-1 was abundant and the only fibrillin isoform. MAGP-1 was the only other microfibril-associated molecule. gamma-Crystallin co-purified with zonular microfibrils, so this association may contribute to ciliary zonule anchorage to lens. Recombinant fibrillin-1 tryptic peptides mapped throughout the molecule and included virtually all predicted peptides except for those larger than 4.5 kDa, smaller than 600 Da or post-translationally modified. In contrast, fewer microfibril tryptic fibrillin-1 peptides were detected, although they were derived from domains throughout the molecule and included two peptides after the C-terminal furin processing site. Several microfibril-derived N- and C-terminal domains never yielded any peptides, while tryptic peptides from other domains yielded numerous peptides, suggesting that some tissue microfibril features are retained after trypsinisation. This first MS analysis of a purified extracellular matrix assembly has provided new insights into microfibril composition and fibrillin-1 organisation within them.

Adult↗
PubMed2006

Semi-supervised LC/MS alignment for differential proteomics.

MOTIVATION: Mass spectrometry (MS) combined with high-performance liquid chromatography (LC) has received considerable attention for high-throughput analysis of proteomes. Isotopic labeling techniques such as ICAT [5,6] have been successfully applied to derive differential quantitative information for two protein samples, however at the price of significantly increased complexity of the experimental setup. To overcome these limitations, we consider a label-free setting where correspondences between elements of two samples have to be established prior to the comparative analysis. The alignment between samples is achieved by nonlinear robust ridge regression. The correspondence estimates are guided in a semi-supervised fashion by prior information which is derived from sequenced tandem mass spectra. RESULTS: The semi-supervised method for finding correspondences was successfully applied to aligning highly complex protein samples, even if they exhibit large variations due to different biological conditions. A large-scale experiment clearly demonstrates that the proposed method bridges the gap between statistical data analysis and label-free quantitative differential proteomics. AVAILABILITY: The software will be available on the website http://people.inf.ethz.ch/befische/proteomics.

Algorithms↗
PubMed2006

Proteomics and metabolomics: the molecular make-up of toxic aromatic pollutant bioremediation.

Microbial-mediated attenuation of toxic aromatic pollutants offers great potential for the restoration of contaminated environments in an ecologically acceptable manner. However, incomplete biological information regarding the regulation of growth and metabolism in many microbial communities restricts progress in the site-specific mineralization process. In the postgenomic era, recent advances in MS have allowed enormous progress in proteomics and elucidated many complex biological interactions. These research forefronts are now expanding toward the analysis of low-molecular-weight primary and secondary metabolites analysis, i.e., metabolomics. The advent of 2-DE in conjunction with MS offers a promising approach to address the molecular mechanisms of bioremediation. The two fields of proteomics and metabolomics have thus far worked separately to identify proteins and primary and secondary metabolites during bioremediation. A simultaneous study combining functional proteomics and metabolomics, i.e., proteometabolomics would create a system-wide approach to studying site-specific microorganisms during active mineralization processes. This article deals with advances in environmental proteomics and metabolomics and advocates the simultaneous study of both technologies to implement cell-free bioremediation.

Biodegradation, Environmental↗
PubMed2000

Proteomics: a powerful tool in the post-genomic era.

Genomics is having a profound impact on biological research, including photosynthesis investigations. Genomes of many photosynthetic organisms have been sequenced. The information about ALL genes that govern and execute photoautotrophic metabolism provides many opportunities to understand genome function and details of known and uncharted pathways. Proteomics, analysis of the protein complement of the genome, is a powerful tool in understanding which proteins are present in a particular tissue under given conditions. Proteomics also allows us to estimate relative levels of proteins and to determine post-translational modifications of the gene products. In this minireview, we discuss the technology and its applications in plant sciences.

Gene Expression Regulation↗