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Results for “differential proteomics”

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PubMed2005

Differential proteomic serum pattern of low molecular weight proteins expressed by adenocarcinoma lung cancer patients.

Based on the assumption that proteins can emanate from tumour to serum, we investigated whether serum low molecular weight proteins (LMW) can discriminate lung cancer patients from healthy donors. Pooled sera from 20 lung cancer patients matched in sex (men), histological type (adenocarcinoma) and stage (IIIB and IV) and from 20 healthy donors (men) were submitted to 2-DE coupled to MALDI-TOF peptide mass fingerprinting. Results of 2D-E/ MALDI-TOF showed five up regulated proteins (immunoglobulin lambda chain, transthyretin monomer, haptoglobin-alfa 2 and two isoforms of serum amyloid protein) and one down regulated (fragment of apolipoprotein A-I) in patients. All differentially expressed proteins, except immunoglobulin, may be acting as a non-specific sign of inflammation in cancer and transthyretin monomer may act as a possible blood marker to CSF barrier disruption that occurs, e.g., in cerebral metastasis. In conclusion, this work shows the usefulness of 2D-gel electrophoresis and mass spectrometry proteomic techniques for the identification of up- and down-regulated proteins in serum from adenocarcinoma lung cancer patients.

Adenocarcinoma↗
PubMed2005

Differential proteome analysis of conditioned media to detect Smad4 regulated secreted biomarkers in colon cancer.

Smad4 is a tumor suppressor gene primarily involved in carcinogenesis of the pancreas and colon. The functional inactivation of Smad4 is a late step genetically. In pancreatic carcinogenesis, loss of Smad4 marks the transition to invasive growth. In colorectal cancers, the frequency of Smad4 inactivation is markedly increased in metastatic cancers. We have established cell biological models, re-expressing Smad4 in deficient human cancer cells, in which we could show that Smad4 is adequate to suppress tumor growth through suppression of angiogenic and invasive properties. Thus, pairs of Smad4-re-expressing and Smad4-deficient cells are prone to model the progression from premalignant stages to carcinomas in the carcinogenic process and may provide access to Smad4 targets of high clinical relevance. We present here a "differential secretome analysis", comparing all the proteins released in vitro from the Smad4-deficient and Smad4-re-expressing SW480 human colon carcinoma cells. The differential secretome catalog comprises more than 25 proteins including proteases and protease inhibitors, as well as established tumor biomarkers. In conclusion, this approach proved to be a sensitive tool to specifically detect Smad4 targets relevant for tumor-stroma interactions. It is also able to reflect complex alterations of cellular physiology. Moreover, the results support our hypothesis that human tumor markers detectable in serum may be identified through differential secretome analyses.

Biomarkers, Tumor↗
PubMed2006

Differential proteome analysis of colon carcinoma cell line SW480 after reconstitution of the tumour suppressor Smad4.

The tumour suppressor gene Smad4 is frequently inactivated in gastrointestinal carcinomas. Smad4 plays a pivotal role in transducing signals of the transforming growth factor-beta (TGF-beta) superfamily of proteins. Inactivation of Smad4 seems to occur late during tumour progression when tumours acquire invasive and metastatic properties. Identification of proteins directly or indirectly regulated by Smad4 would, therefore, ease the future design of new diagnostic and therapeutic strategies for gastrointestinal carcinoma. We have used human colon carcinoma cell line SW480 stably transfected with Smad4 as an in-vitro model system to identify Smad4-regulated proteins by applying two-dimensional gel electrophoresis (2DE) then MALDI-PMF/PFF-MS. We identified a total of 47 protein species with a Smad4-dependent expression. From the functions of the candidate proteins we obtained new insights into Smad4's participation in processes, for example apoptosis, differentiation, and proliferation.

Amino Acid Sequence↗
PubMed2003

Differential proteomic analysis of the mouse retina: the induction of crystallin proteins by retinal degeneration in the rd1 mouse.

We have applied proteomic analysis to the degeneration of photoreceptors. In the rd1 mouse, a recessive mutation in the PDE6B gene leads to rapid loss of rods through apoptosis. By 5 wk postnatal, virtually all rod photoreceptors have degenerated, leaving one row of cones that degenerates secondarily. In order to assess comparative protein expression, proteins extracted from whole retina were resolved on a two-dimensional gel and identified by mass spectrometry combined with database screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry coupled to peptide mass fingerprinting was sufficient to identify most of the proteins, the remaining being identified with additional sequence information obtained by nano-electrospray ionization tandem mass spectrometry or liquid chromatography tandem mass spectrometry. The study revealed 212 spots, grouped into 109 different proteins. Differential analysis showed loss of proteins involved in the rod-specific phototransduction cascade, as well as induction of proteins from the crystallin family, in response to retinal degeneration. Identification of such pathways may contribute to new therapeutic approaches.

Amino Acid Sequence↗
PubMed2006

Differential proteomic analysis of the Bacillus anthracis secretome: distinct plasmid and chromosome CO2-dependent cross talk mechanisms modulate extracellular proteolytic activities.

The secretomes of a virulent Bacillus anthracis strain and of avirulent strains (cured of the virulence plasmids pXO1 and pXO2), cultured in rich and minimal media, were studied by a comparative proteomic approach. More than 400 protein spots, representing the products of 64 genes, were identified, and a unique pattern of protein relative abundance with respect to the presence of the virulence plasmids was revealed. In minimal medium under high CO(2) tension, conditions considered to simulate those encountered in the host, the presence of the plasmids leads to enhanced expression of 12 chromosome-carried genes (10 of which could not be detected in the absence of the plasmids) in addition to expression of 5 pXO1-encoded proteins. Furthermore, under these conditions, the presence of the pXO1 and pXO2 plasmids leads to the repression of 14 chromosomal genes. On the other hand, in minimal aerobic medium not supplemented with CO(2), the virulent and avirulent B. anthracis strains manifest very similar protein signatures, and most strikingly, two proteins (the metalloproteases InhA1 and NprB, orthologs of gene products attributed to the Bacillus cereus group PlcR regulon) represent over 90% of the total secretome. Interestingly, of the 64 identified gene products, at least 31 harbor features characteristic of virulence determinants (such as toxins, proteases, nucleotidases, sulfatases, transporters, and detoxification factors), 22 of which are differentially regulated in a plasmid-dependent manner. The nature and the expression patterns of proteins in the various secretomes suggest that distinct CO(2)-responsive chromosome- and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.

Amino Acid Sequence↗
PubMed2004

Differential proteomic analysis of Aeromonas salmonicida outer membrane proteins in response to low iron and in vivo growth conditions.

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis, a serious infectious disease of salmonids. Bacterial phenotypes are known to change in vivo compared to the in vitro state. Proteomic analysis of in vivo phenotypes is usually not possible due to insufficient biomass. Using an in vivo growth chamber model, the pathogenic fish bacterium A. salmonicida was cultured in pure culture in vivo in its host, the Atlantic salmon, to obtain sufficient biomass to allow proteomic analysis. Growth of A. salmonicida under in vitro iron-restricted conditions resulted in the expression of outer membrane proteins of 73, 76 and 85 kDa, which were not present when grown under in vitro iron-replete conditions. Mass spectrometry analysis identified the 73 kDa protein as a colicin receptor, the 76 kDa protein as an outer membrane heme receptor, and the 85 kDa protein as a ferric siderophore receptor. When cultured in vivo, A. salmonicida up-regulated the identical 73, 76 and 85 kDa proteins. The results of this study also suggest, at least with respect to the outer membrane proteins, that the in vitro iron-restricted growth model largely reproduces the results obtained from growth of A. salmonicida within the peritoneal cavity of salmon.

Aeromonas↗
PubMed2004

Differential proteome analysis and mass spectrometric characterization of germ line development-related proteins of Caenorhabditis elegans.

Proteome maps and differences of protein patterns of the synchronized larval stage L4 of the temperature-sensitive Caenorhabditis elegans (C. elegans) glp-1 mutant (e2144ts) were investigated after cultivation at 15 degrees C (developing a normal phenotype) or 25 degrees C (developing a mutated phenotype) by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. From the 183 identified protein spots six proteins were found differently expressed. The Vit-6 vitellogenin (CE28594), the hypothetical 17.2 protein (CE25224), the hypothetical 17.4 protein (CE16999), and the heat shock protein 16 kDa (CE14249) were more abundant when growing worm cultures at 25 degrees C. By contrast, the nucleoside diphosphate kinase (CE09650) was found increased at 15 degrees C. Most notably, the eukariotic initiation factor 5A-1 (CE00503), highly abundant at 15 degrees C, was not present in cultures grown at 25 degrees C. Its absence at 25 degrees C can not be attributed to lack of the enzymatic machinery that is necessary for hypusinylation. Instead, a direct downstream effect of the lack of functionality of GLP-1 may cause the expression of this protein. The yolk proteins 115 kDa and 88 kDa were attributed by mass spectrometric protein structure analysis as C-terminal and N-terminal fragments of the Vit-6 vitellogin protein (CE28594), respectively. The cleavage site between both derivatives was located between R764 and A768. A conflict in the database sequences at amino acid positions 1622 and 1623 of vitellogenin-6 was solved by mass spectrometric sequence analysis. The combination of 2-DE with mass spectrometry enabled the identification of mutation-associated differences on somatic gonadal cell and germ line cell development-associated proteins.

Amino Acid Sequence↗
PubMed2005

A differential proteome in tumors suppressed by an adenovirus-based skin patch vaccine encoding human carcinoembryonic antigen.

We created an anti-tumor vaccine by using adenovirus as a vector which contains a cytomegalovirus early promoter-directed human carcinoembryonic antigen gene (AdCMV-hCEA). In an attempt to develop the skin patch vaccine, we epicutaneously vaccinated Balb/c mice with AdCMV-hCEA. After nine weeks post-immunization, vaccinated mice evoked a robust antibody titer to CEA and demonstrated the capability of suppressing in vivo growth of implanted murine mammay adenocarioma cell line (JC-hCEA) tumor cells derived from a female Balb/c mouse. Proteomic analysis of the tumor masses in the non-vaccinated naive and vaccinated mice reveal that six proteins change their abundance in the tumor mass. The levels of adenylate kinase 1, beta-enolase, creatine kinase M chain, hemoglobin beta chain and prohibitin were statistically increased whereas the level of a creatine kinase fragment, which is undocumented, was decreased in the tumor of vaccinated mice. These proteins may provide a vital link between early-stage tumor suppression and immune response of skin patch vaccination.

Adenoviridae↗
PubMed2005

Differential proteomic analysis of proteins in wheat spikes induced by Fusarium graminearum.

Scab, caused by Fusarium graminearum, is a serious spike disease in wheat. To identify proteins in resistant wheat cultivar Wangshuibai induced by F. graminearum infection, proteins extracted from spikes 6, 12 and 24 h after inoculation were separated by 2-DE. Thirty protein spots showing 3-fold change in abundance when compared with treatment without inoculation were characterized by MALDI-TOF MS and matched to proteins by querying the mass spectra in protein databases or the Triticeae EST translation database. Based on their volume profiles, these proteins were classified into four categories. The first one fell off rapidly at the initial inoculation and then rose at 12 or 24 hai, the second one decreased considerably after inoculation and remained at low level, the third one rose at the initial inoculation and then declined at 12 or 24 hai, the forth one showed steady increase after inoculation and maintained at a high level. Many of the proteins identified in the first two categories are related to carbon metabolism and photosynthesis. While most of proteins identified in the last two categories are related to stress defense of plants, indicating that proteins associated with the defense reactions were activated or translated shortly after inoculation.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Differential proteomic analysis of the anti-proliferative effect of glucocorticoid hormones in ST1 rat glioma cells.

Glucocorticoid hormones (GCs) exert a potent anti-proliferative activity on several cell types. The classic molecular mechanism of GCs involves modulation of the activity of the glucocorticoids receptor, a transcriptional regulator. However, the anti-proliferative effect of GCs may also involve modulation of processes such as translation, subcellular localization and post-translational modifications, which are not reflected at the mRNA level. To investigate these potential effects of GCs, we employed the proteomic approach (two-dimensional electrophoresis and mass spectrometry) and the ST1 cells, obtained from the C6 rat glioma cell line, as a model. GC treatment leads ST1 cells to a complete transformed-to-normal phenotypic reversion and loss of their tumorigenic potential. By comparing sets of 2D nuclear protein profiles of ST1 cells treated (or not) with hydrocortisone (Hy), 13 polypeptides displaying >or=two-fold difference in abundance upon Hy treatment were found. Five of these polypeptides were identified by peptide mass fingerprinting, including Annexin 2 (ANX2), hnRNP A3 and Ubiquitin. Evidence obtained by Western blot analysis indicates that ANX2 is present in the nucleus and has its subcellular localization modulated by GC-treatment of ST1 cells. Our findings indicate complementary mechanisms contributing to the regulation of gene expression associated with ST1 cells' response to GCs.

Animals↗
PubMedDate not supplied

Differential proteomic analysis reveals increased cathelicidin expression in porcine bronchoalveolar lavage fluid after an Actinobacillus pleuropneumoniae infection.

Accurate definition of respiratory health in pigs is an important problem for swine producers and veterinarians. In an approach to identify potential biomarkers, two-dimensional gel electrophoresis and mass spectrometry on bronchoalveolar lavage fluid (BALF)-derived proteins from pigs experimentally infected with Actinobacillus pleuropneumoniae were performed at different time points post infection. Mock-infected pigs were used as a control. It was shown that the antimicrobial peptides, prophenin-2 and PR-39, and the calcium-binding protein calgranulin C were reproducibly upregulated in BALF of pigs chronically infected with A. pleuropneumoniae. Concentrations of PR-39 were significantly (p<0.05) increased in BALF (median of 4.8 nM) but not in serum (median of 2.5 nM) on day 21 after infection. A Receiver Operating Characteristics (ROC) plot showed that PR-39 in BALF is an accurate and easily accessible marker to detect clinically healthy pigs convalescent from an experimental A. pleuropneumoniae infection. These results imply that PR-39 might have a potential as a general biomarker to determine porcine respiratory health.

Actinobacillus Infections↗
PubMed2003

Differential proteome analysis: two-dimensional nano-LC/MS of E. coli proteome grown on different carbon sources.

Different sugars provided to bacteria as single sources of carbon and energy require the induction of different metabolic enzymes, transporters, and uptake systems in order to support growth and cell survival. Using a nano-high-performance liquid chromatography/mass spectrometry (nano-HPLC/MS) system we constructed comprehensive peptide maps for Escherichia coli grown with either lactose or glucose in minimal medium. Digested bacterial samples were separated in a two-dimensional manner by combining strong cation exchange (SCX) and reversed-phased (RP) chromatography. Peptides were eluted online to an iontrap MS instrument and further analyzed by tandem MS fragmentation. Bacterial proteins originating from the differing samples were analyzed by searching the Swiss Prot Database. Data are presented that show the ability to detect several hundred different proteins significantly expressed under both conditions. Several enzymes and binding proteins related to the lactose metabolism were only identified in the sample grown with this carbon source.

Carbon↗
PubMed2005

Differential proteomic analysis in the study of prokaryotes stress resistance.

Proteomic technologies are powerful tools to study the physiological response of bacteria to various environmental stress conditions. Complex protein toolkits coordinated by regulatory networks have evolved to accommodate bacterial survival under several stressing conditions, such as varying temperature or pH, and changes in nutrient availability. This manuscript provides an overview of the major achievements obtained by 2D gel based and non-gel based proteomic approaches for the study of adaptive response in bacteria. As an example, a study carried out on Streptococcus thermophilus response to various stresses is here reported. In this investigation, we used combined proteomic strategies to define proteomic maps useful to highlight protein profile variations following environmental challenges.

Adaptation, Physiological↗
PubMed2006

Differential serum proteomic analysis in a model of metabolic disease.

Protein profiling would aid in better understanding the pathophysiology of metabolic disease. Here, we report on differential proteomic analysis using an animal model of diabetes mellitus and associated metabolic disorders (Otsuka Long-Evans Tokushima Fatty rat). Serum was analyzed by a new two-dimensional liquid chromatography system which separated proteins by chromatofocusing and subsequent reversed-phase chromatography. This is the first application of this approach to differential serum proteomics. Differentially expressed proteins, identified with MALDI-TOF mass spectrometry, included apolipoproteins and alpha2-HS-glycoprotein. These findings add to our understanding of the underlying pathophysiology. This new proteomic analysis is a promising tool to elucidate disease mechanisms.

Animals↗
PubMedDate not supplied

Differential detergent fractionation for non-electrophoretic eukaryote cell proteomics.

Differential detergent fractionation (DDF), which relies on detergents to sequentially extract proteins from eukaryotic cells, has been used to increase proteome coverage of 2D-PAGE. Here, we used DDF extraction in conjunction with the nonelectrophoretic proteomics method of liquid chromatography and electrospray ionization tandem mass spectrometry. We demonstrate that DDF can be used with 2D-LC ESI MS2 for comprehensive cellular proteomics, including a large proportion of membrane proteins. Compared to some published methods designed to isolate membrane proteins specifically, DDF extraction yields comprehensive proteomes which include twice as many membrane proteins. Two-thirds of these membrane proteins have more than one trans-membrane domain. Since DDF separates proteins based upon their physicochemistry and subcellular localization, this method also provides data useful for functional genome annotation. As more genome sequences are completed, methods which can aid in functional annotation will become increasingly important.

Animals↗
PubMed2004

Proteomic analysis of adipocyte differentiation: Evidence that alpha2 macroglobulin is involved in the adipose conversion of 3T3 L1 preadipocytes.

Adipogenesis is an important aspect of energy homeostasis. Here we have used a differential proteome mapping strategy to identify intracellular proteins that are differentially expressed during adipose conversion of 3T3 L1 preadipocytes. Two-dimensional gel electrophoresis analysis identified 8 proteins that are induced following hormone-evoked differentiation. In addition, we found that a alpha2 macroglobulin fragment was abundantly present in 3T3 L1 preadipocytes, but was virtually undetectable in fully differentiated adipocytes. Metabolic radiolabeling with (35S)methionine and Northern blot analysis indicated that the intracellular alpha2 macroglobulin fragment in preadipocytes was derived from the extracellular culture medium, not de novo synthesis. Incubation of preadipocytes with an antialpha2 macroglobulin polyclonal antibody caused depletion of the intracellular alpha2 macroglobulin fragments, and also enhanced spontaneous adipose conversion. These results suggest that intracellular alpha2 macroglobulin fragment inhibits adipocyte differentiation, and that hormone treatment induces differentiation at least in part by suppression of intracellular alpha2 macroglobulin activity in 3T3 L1 preadipocytes.

3T3 Cells↗
PubMed2001

Proteomic analysis of proteins in PC12 cells before and after treatment with nerve growth factor: increased levels of a 43-kDa chromogranin B-derived fragment during neuronal differentiation.

Proteomic analysis is an important approach to characterizing the proteome and studying protein function in the post-genomic era. It is also a powerful screening method for detecting unexpected alterations in protein expression that may be missed by conventional biochemical techniques. The aim of this study was to perform a preliminary proteomic analysis of PC12 cells in order to investigate the effect of nerve growth factor (NGF) on protein expression in PC12 cells during neurite outgrowth. PC12 cell proteins were separated by two-dimensional electrophoresis (2DE) and visualized by silver staining, then certain proteins were identified by N-terminal amino acid microsequencing and a homology search of a protein sequence database. Over 400 proteins were detected, 10% of which showed a significant (greater than 30%) increase or decrease in expression during NGF-induced neuronal differentiation. Seven proteins in the 2DE map were identified; the levels of five of these were unaffected by NGF treatment, whereas the levels of the other two, beta-tubulin and a novel 43-kDa chromogranin B-derived fragment, were significantly increased by more than 30 and 200%, respectively. Our results suggest that chromogranin B processing is enhanced in PC12 cells during NGF-induced neuronal differentiation. In addition, since this increase in the levels of the chromogranin B-derived fragment was specifically blocked by PD98059, we suggest that the increased processing can be ascribed to activation of the MAP kinase pathway, and that the 43-kDa chromogranin B-derived fragment can serve as a new marker of neuronal differentiation for proteomic studies.

Animals↗