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At least 253 records · Page 14Linked to original sources
PubMed2005

Eye lens proteomics: from global approach to detailed information about phakinin and gamma E and F crystallin genes.

Exploration of the lenticular proteome poses a challenging and worthwhile undertaking as cataracts, the products of a disease phenotype elicited by this proteome, remains the leading cause of vision impairment worldwide. The complete ten day old lens proteome of Mus musculus C57BL/6J was resolved into 900 distinct spots by large gel carrier ampholyte based 2-DE. The predicted amino acid sequences of all 16 crystallins ubiquitous in mammals were corroborated by mass spectrometry (MS). In detailed individual spot analyses, the primary structure of the full murine C57BL/6J beaded filament component phakinin CP49 was sequenced by liquid chromatography/electrospray ionization-tandem MS and amended at two positions. This definitive polypeptide sequence was aligned to the mouse genome, thus identifying the entire C57BL/6J genomic coding region. Also, two murine C57/6J polypeptides, both previously classified as gamma F crystallin, were clearly distinguished by MS and electrophoretic mobility. Both were assigned to their respective genes, one of the polypeptides was reclassified as C57BL/6J gamma E crystallin. Building on these data and previous investigations an updated crystallin reference map was put forth and several non crystallin lenticular components were examined. These results represent the first part of a comprehensive investigation of the mouse lens proteome (http://www.mpiib-berlin.mpg.de/2D-PAGE) with emphasis on understanding genetic effects on proteins and disease development.

Amino Acid Sequence↗
PubMed2003

Proteome analysis on an early transformed human bronchial epithelial cell line, BEP2D, after alpha-particle irradiation.

To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.

Alpha Particles↗
PubMed2003

Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line.

A proteomics approach was used to identify the proteins potentially implicated in the cellular response concomitant with elevated production levels of human growth hormone in a recombinant Chinese hamster ovary (CHO) cell line following exposure to 0.5 mM butyrate and 80 microM zinc sulphate in the production media. This involved incorporation of two-dimensional (2-D) gel electrophoresis and protein identification by a combination of N-terminal sequencing, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, amino acid analysis and cross species database matching. From these identifications a CHO 2-D reference map and annotated database have been established. Metabolic labelling and subsequent autoradiography showed the induction of a number of cellular proteins in response to the media additives butyrate and zinc sulphate. These were identified as GRP75, enolase and thioredoxin. The chaperone proteins GRP78, HSP90, GRP94 and HSP70 were not up-regulated under these conditions.

Amino Acids↗
PubMed2005

Profiling of abundant proteins associated with dichlorodiphenyltrichloroethane resistance in Drosophila melanogaster.

Dichlorodiphenyltrichloroethane (DDT) metabolism-based resistance in Drosophila melanogaster is a complex metabolic system associated with the transcription of detoxification related genes, ion transport, lipid and sugar metabolism pathways. However, little is known about the differences regarding the proteome of field- and laboratory-selected resistant Drosophila genotypes. We investigated the impact of DDT resistance in the abundant proteome of field- and laboratory- selected resistant Drosophila using a two-dimensional gel electrophoresis DDT reference map. Proteomic profiling was performed in two DDT susceptible genotypes (Canton-S and 91-C) and three DDT resistant lines (Rst(2)DDT(91-R), Rst(2)DDT(Wisconsin) and Rst(2)DDT(Hikone-R)). Protein spots were stained with Coomassie blue and compared using PDQuest software. Selected protein spots were cut out and analyzed using matrix assisted laser desorption-time of flight mass spectrometry. Querying the NCBInr. 10.21.2003 database with mass spectrometric data yielded the identity of 21 differentially translated proteins in Rst(2)DDT(91-R), Rst(2)DDT(Wisconsin) and Canton-S representing proteins putatively involved in biochemical pathways such as glycolysis and gluconeogenesis, the pentose phosphate pathway, the Krebs cycle and fatty acid oxidation. We hypothesize that both strategies are aimed to use of the pentose phosphate pathway to increase glucose utilization while Rst(2)DDT(91-R) relies primarily on glycolysis to produce reduced NADP and increase DDT detoxification. DDT exposure in Canton-S induced six proteins, while four proteins were repressed in Rst(2)DDT(Hikone-R). Our data suggest that insecticide resistance appears to impact different metabolic pathways in Drosophila genotypes selected with the same pesticide (DDT).

Animals↗
PubMed2004

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of the bacterial pathogen Bartonella henselae.

Bartonella henselae is an emerging zoonotic pathogen causing a wide range of disease manifestations in humans. In this study, we report on the analysis of the sarcosine-insoluble outer membrane fraction of B. henselae ATCC 49882 Houston-1 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) and two-dimensional nonequilibrium pH gradient polyacrylamide gel electrophoresis (2-D NEPHGE). Protein species were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and subsequent database query against the B. henselae genome sequence. Subcellular fractionation, application of the ionic detergent lauryl sarcosine, assessment of trypsin sensitivity, and heat modifiability of surface-exposed proteins represented valuable tools for the analysis of the outer membrane subproteome of B. henselae. 2-D NEPHGE was applied to display and catalogue a substantial number of proteins associated with the B. henselae sarcosine-insoluble outer membrane fraction, resulting in the establishment of a first 2-D reference map of this compartment. Thus, 53 distinct protein species associated with the outer membrane subproteome fraction were identified. This study provides novel insights into the membrane biology and the associated putative virulence factors of this pathogen of increasing medical importance.

Bacterial Proteins↗
PubMed2005

The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins.

The lysosome is a membrane delimited cytoplasmic organelle that contains at least 50 hydrolytic enzymes and associated cofactors. The biomedical importance of these enzymes is highlighted by the many lysosomal storage disorders that are associated with mutations in genes encoding lysosomal proteins, and there is also evidence that lysosomal activities may be involved in more widespread human diseases. The aim of this study was to characterize the human brain lysosomal proteome with the goal of establishing a reference map to investigate human diseases of unknown etiology and to gain insights into the cellular function of the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified using immobilized Man6-P receptor. Fractionation by two-dimensional electrophoresis resolved a complex mixture comprising approximately 800 spots. Constituent proteins in each spot were identified using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (both peptide mass fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic digests and N-terminal sequencing. In a complementary analysis, we also analyzed a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins. Forty-one were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. An additional nine proteins were either uncharacterized or proteins not previously reported to have lysosomal function. We found that the human brain Man6-P-containing lysosomal proteome is highly complex and contains more proteins with a much greater number of individual isoforms than found in previous studies of Man6-P glycoproteomes.

Brain↗
PubMed2005

Francisella tularensis live vaccine strain: proteomic analysis of membrane proteins enriched fraction.

Proteome analysis of Gram-negative facultative intracellular pathogen Francisella tularensis (F. tularensis) live vaccine strain has been performed only on whole-cell extracts so far. This is the first study dealing with the analysis of the membrane subproteome of this microorganism. A fraction enriched in membrane proteins obtained by carbonate extraction was separated using two-dimensional electrophoresis and all visualized spots were identified by mass spectrometry. The reference map is the basis for further comparative analyses of virulent and non-virulent F. tularensis strains.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Proteomic analysis of neural differentiation of mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) can differentiate into different types of cells, and serve as a good model system to study human embryonic stem cells (hESCs). We showed that mESCs differentiated into two types of neurons with different time courses. To determine the global protein expression changes after neural differentiation, we employed a proteomic strategy to analyze the differences between the proteomes of ES cells (E14) and neurons. Using 2-DE plus LC/MS/MS, we have generated proteome reference maps of E14 cells and derived dopaminergic neurons. Around 23 proteins with an increase or decrease in expression or phosphorylation after differentiation have been identified. We confirmed the downregulation of translationally controlled tumor protein (TCTP) and upregulation of alpha-tubulin by Western blotting. We also showed that TCTP was further downregulated in derived motor neurons than in dopaminergic neurons, and its expression level was independent of extracellular Ca(2+) concentration during neural differentiation. Potential roles of TCTP in modulating neural differentiation through binding to Ca(2+), tubulin and Na,K-ATPase, as well as the functional significance of regulation of other proteins such as actin-related protein 3 (Arp3) and Ran GTPase are discussed. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.

Animals↗
PubMed2005

Proteomic analysis of the response of human lung cells to uranium.

The industrial use of uranium and particularly of depleted uranium, has pinpointed the need to review its chemical impact on human health. A proteomic approach was used to evaluate the response of a human lung cell line (A549) to uranium. We established the first 2-D reference map of the A549 cell line, identifying 87 spots corresponding to 81 major proteins. Uranium treatment triggered differential expression of 18 spots, of which 14 corresponded to fragments of cytokeratin 8 (CK8) and cytokeratin (CK18) and 1 to peroxiredoxin 1. We probed several hypotheses regarding CK cleavage, and observed that it did not result from caspase or calpain activity. Furthermore, we showed that the fragments are recognised by an anti-ubiquitin antibody (KM691). These results suggest a regulatory pathway involving CK ubiquitinylation or dysfunction in the proteasome-ubiquitin system in response to uranium exposure in human lung cells.

Amino Acid Sequence↗
PubMed2005

Proteomic characterization of human normal articular chondrocytes: a novel tool for the study of osteoarthritis and other rheumatic diseases.

Articular cartilage is composed of cells and an extracellular matrix. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. There is currently a great lack of knowledge about the chondrocyte proteome. To solve this deficiency, we have obtained the first reference map of the human normal articular chondrocyte. Cells were isolated from cartilages obtained from autopsies without history of joint disease. Cultured cells were used to obtain protein extracts which were resolved by 2-DE and visualized by silver nitrate or CBB staining. Almost 200 spots were excised from the gels and analyzed using MALDI-TOF or MALDI-TOF/TOF MS. The analysis leads to the identification of 136 spots that represent 93 different proteins. A significant proportion of proteins are involved in cell organization (26%), energy (16%), protein fate (14%), metabolism (12%), and cell stress (12%). From all the identified proteins, annexins, vimentin, transgelin, destrin, cathepsin D, heat shock protein 47, and mitochondrial superoxide dismutase were more abundant in chondrocytes than in other types of mesenchymal cells such as Jurkat-T cells. As metabolic program of chondrocytes is altered in osteoarthritis and other rheumatic diseases, this proteomic map is an important tool for future studies on these pathologies.

Adolescent↗
PubMed2006

A study of Streptococcus thermophilus proteome by integrated analytical procedures and differential expression investigations.

Streptococcus thermophilus is a Gram-positive bacterium belonging to the group of lactic acid bacteria, among which several genera play an essential role in manufacture of food products. Recently, a genomic consortium sequenced and annotated its entire genome, which has been demonstrated to contain 1900 coding sequences. In this study, we have revealed the expression products of almost 200 different genes using a proteomic strategy combining 2-DE plus MALDI-TOF PMF and differential 1-DE plus muLC-ESI-IT-MS/MS. Thus, a number of cellular pathways related to important physiological processes were described at the proteomic level. Almost 50 genes were related to multiple electrophoretic species, whose heterogeneity was mainly due to variability in pI values. A 2-DE reference map obtained for lactose-grown cells was compared with those obtained after heat, cold, acid, oxidative and starvation stresses. Protein up/down-regulation measurements demonstrated that adaptation to different environmental challenges may involve the contribution of unique as well as combined physiological mechanisms. Common regulatory sites in the promoter region of genes whose expression was induced after stress were identified. These results provide a better comprehension of biochemical processes related to stress resistance in S. thermophilus, allowing defining the molecular bases of adaptative responses or markers for the identification of strains with potential industrial applications.

Bacterial Proteins↗
PubMed2006

Detection of phosphorylation patterns in rat cortical neurons by combining phosphatase treatment and DIGE technology.

Although protein phosphorylation is probably the most studied post-translational modification occurring in cells, the number of proteins, which are the target of this modification, is still largely unknown. Increasing the coverage of the phosphoproteome as well as the detection of variation at the phosphorylation level would be very helpful for understanding the mechanisms of cell life and the modifications of the cell state leading to pathological conditions such as neurodegeneration. In order to further investigate variations occurring at the phosphorylation level, we have initiated the creation of a reference map of phosphorylated proteins in rat cortical neurons, employing a combination of phosphatase treatment and 2-DE/differential in gel electrophoresis technology. About 131 spots were recognized as phosphorylated proteins as they showed different migration behaviour after phosphatase treatment. The analysis of 42 selected spots was carried out by LC/MS/MS technology resulting in the identification of two new phosphoproteins.

Animals↗
PubMed2006

The phosphate-starvation response in Vibrio cholerae O1 and phoB mutant under proteomic analysis: disclosing functions involved in adaptation, survival and virulence.

A proteomic analysis of a wild-type and of a phoB mutant showed that Vibrio cholerae expresses genes of two major regulons in response to phosphate starvation. The Pho regulon, expressed by the wild-type, allowed the cells to adapt to the new environment. Induction of the general stress regulon was mainly observed in the phoB mutant as a strategy to resist stress and survive. Some functions of the adaptative and survival responses play roles in the pathogenicity of the bacteria. Among the members of the Pho regulon, we found a porin described as an important factor for the intestinal colonisation. Other functions not obviously related to phosphate metabolism, expressed preferentially by the wild-type cells, have also been implicated in virulence. These findings might explain the lack of virulence of the phoB mutant. The Pho regulon picture of V. cholerae, however, will not be complete until minor members and membrane proteins are identified. Among the phosphate-starvation induced genes we have found 13 hypothetical ones and for some of them functions have been assigned. The majority of the genes identified here have not been described before, thus they could be used to expand the proteomic reference map of V. cholerae El Tor.

Adaptation, Physiological↗
PubMed2006

Analysis of chicken serum proteome and differential protein expression during development in single-comb White Leghorn hens.

Serum is believed to harbor thousands of distinct proteins that are either actively secreted or leak from various blood cells or tissues. Exploring protein composition in serum may accelerate the discovery of novel protein biomarkers for specific economic traits in livestock species. This study analyzed serum protein composition to establish a 2-DE reference map, and monitored protein dynamics of single-comb White Leghorn hens at 8, 19 and 23 weeks after hatching. A total of 119 CBB-stained and 315 silver-stained serum protein spots were analyzed by MALDI-TOF MS. Of these, 98 CBB-stained and 94 silver-stained protein spots were significantly matched to existing chicken proteins. The identified spots represented 30 distinctive proteins in the serum of laying hens. To compare protein expression during development, expression levels of 47 protein spots were quantified by relative spot volume with Melanie 3 software. Ten protein spots increased and 3 protein spots decreased as hen age increased. Previous research has suggested that some of these proteins play critical roles in egg production. The differentially expressed proteins with unknown identities will be valuable candidates for further explorations of their roles in egg production of laying hens.

Age Factors↗
PubMed2006

Proteomic dissection of plant development.

Plant development is controlled by complex endogenous genetic programs and responses to environmental cues. Proteome analyses have recently been introduced to plant biology to identify proteins instrumental in these developmental processes. To date most plant proteome studies have been employed to generate reference maps of the most abundant soluble proteins of plant organs at a defined developmental stage. However, proteomics is now also utilized for genetic studies comparing the proteomes of different plant genotypes, for physiological studies analyzing the influences of exogenous signals on a particular plant organ, and developmental studies investigating proteome changes during development. Technical advances are now beginning to allow a proteomic dissection of individual cell types, thus greatly increasing the information revealed by proteome analyses.

Plant Development↗
PubMed2006

Comparative proteomics of high light stress in the model alga Chlamydomonas reinhardtii.

High light (HL) stress adversely affects growth, productivity and viability of photosynthetic organisms. The green alga Chlamydomonas reinhardtii is a model system to study photosynthesis and light stress. Comparative proteomics of wild-type and two very high light (VHL)-resistant mutants, VHL(R)-S4 and VHL(R)-S9, revealed complex alterations in response to excess light. A two-dimensional reference map of the soluble subproteome was constructed representing about 1500 proteins. A total of 83 proteins from various metabolic pathways were identified by peptide mass fingerprinting. Quantitative comparisons of 444 proteins showed 105 significantly changed proteins between wild type and mutants under different light conditions. Commonly, more proteins were decreased than increased, but different proteins were affected in each genotype. Proteins uniquely altered in either VHL(R) mutant may be involved in VHL resistance. Such candidate proteins similarly altered without light stress, thus possibly contributing to "pre-adaptation" of mutants to VHL, included decreased levels of a DEAD box RNA helicase (VHL(R)-S4) and NAB1 and RB38 proteins (VHL(R)-S9), and increased levels of an oxygen evolving enhancer 1 (OEE1) isoform and an unknown protein (VHL(R)-S4). Changes from increased levels in HL to decreased levels in excess light, included OEE1 (VHL(R)-S9) or the reverse change for NAB1, RB38, beta-carbonic anhydrase and an ABC transporter-like protein (VHL(R)-S4).

Animals↗
PubMed2006

Proteome analysis of B-cell maturation.

Proteins affected by anti-mIgM stimulation during B-cell maturation were identified using 2-DE-based proteomics. We investigated the proteome profiles of stimulated and nonstimulated Ramos B-cells at eight time points during 5 d and compared the obtained proteomic data to the corresponding data from DNA-microarray studies. Anti-mIgM stimulation of the cells resulted in significant differences (> or =twofold) in the protein abundance close to 100 proteins and differences in post-translational protein modifications. Forty-eight up- or down-regulated proteins were identified by mass spectrometric methods and database searches. The identities of a further nine proteins were revealed by comparing their positions to the known proteins in other lymphocyte 2-DE databases. Several of the proteins are directly related to the functional and morphological characteristics of B-cells, such as cytoskeleton rearrangement and intracellular signalling triggered by the crosslinking of B-cell receptors. In addition to proteins known to be involved in human B-cell maturation, we identified several proteins that were not previously linked to lymphocyte differentiation. The results provide deeper insights into the process of B-cell maturation and may lead to novel therapeutic strategies for immunodeficiencies. An interactive 2-DE reference map is available at http://bioinf.uta.fi/BcellProteome.

B-Lymphocytes↗
PubMed2006

Rejuvenating rice proteomics: facts, challenges, and visions.

Proteomics is progressing at an unprecedented pace, as can be exemplified by the progress in model organisms such as yeast, bacteria, and mammals. Proteomics research in plants, however, has not progressed at the same pace. Unscrambling of the genome sequences of the dicotyledoneous Arabidopsis thaliana (L.) and monocotyledoneous rice (Oryza sativa L.) plant species, respectively, has made them accessible reference organisms to study plant proteomics. Study of these two reference plants is expected to unravel the mystery of plant biology. Rice, a critically important food crop on the earth, has been termed a "cornerstone" and the "Rosetta stone" for functional genomics of cereal crops. Here, we look at the progress in unraveling rice proteomes and present the facts, challenges, and vision. The text is divided into two major parts: the first part presents the facts and the second part discusses the challenges and vision. The facts include the technology and its use in developing proteomes, which have been critically and constructively reviewed. The challenges and vision deal with the establishment of technologies to exhaustively investigate the protein components of a proteome, to generate high-resolution gel-based reference maps, and to give rice proteomics a functional dimension by studying PTMs and isolation of multiprotein complexes. Finally, we direct a vision on rice proteomics. This is our third review in series on rice proteomics, which aims to stimulate an objective discussion among rice researchers and to understand the necessity and impact of unraveling rice proteomes to their full potential.

Arabidopsis↗