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PubMed2006

Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis.

Normal Chinese Liver Proteome Expression Profile is one of the major parts of Human Liver Proteome Project. Before starting the studies, it is necessary to examine the interindividual variation of normal liver proteome and evaluate the minimal size of samples for proteomic analysis. In this study, normal liver samples from ten individual volunteers were collected and the proteome profiles of these samples were analyzed using 2-D difference gel electrophoresis (DIGE) combined with MALDI-TOF/TOF MS. The individual liver tissue lysates were labeled with Cy3 and Cy5 while the pooled sample was labeled with Cy2 as an internal standard, which minimized gel-to-gel variation. After analysis by the DeCyder software, up to 2056 protein spots were detected on the master gel. The CV of standardized abundance was calculated for the protein spots that were matched across all ten gels. The CV values of these protein spots ranged from 6.4 to 108.5% and the median CV was approximately 19%, which demonstrated that the protein expression of normal liver among different individuals was relatively stable. The eight proteins with CV values over 50% were identified which would be a caveat when considering these proteins as potential disease-related markers. Moreover, the one-way ANOVA feature showed a correlation between sample size and individual variations. The results showed that when the sample size exceeded 7, the individual variations were not significant to the whole pool. Our results are an important basis for liver protein expression profiles and comparative proteomics of liver disease.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Proteomic analysis of Rickettsia prowazekii.

Rickettsia prowazekii is an obligate intracellular gram-negative bacterium. Comparative proteomics study of a virulent strain (Breinl) versus an avirulent strain (Madrid E) was performed using an integrated liquid chromatography and mass spectrometer. About 30% of predicted proteins were detected and identified. Among the detected proteins, more than 30 proteins were of unknown function in both strains. Although several proteins were detected in only one strain, the overall distribution of detected proteins in different COGs (clusters of orthologs groups) was very similar between the two strains. Functional analysis of differentially expressed proteins, either qualitatively or quantitatively, may lead to the discovery of pathogenesis-related factors.

Bacterial Proteins↗
PubMed2006

A proteomic analysis of protein variations during differentiation of v-myb-transformed monoblasts.

v-myb oncogene of avian myeloblastosis virus (AMV) transforms myelomonocytic cells in vitro and induces acute monoblastic leukemia in vivo. The transforming effect of the v-myb can be suppressed using phorbol ester (TPA) or histone deacetylase inhibitor trichostatin A (TSA), the inducers of cell differentiation that are in clinical trials. In this study, we used proteomics-based approach to identify proteins with variable expression in differentiated BM2 cells. Proteome variations induced by TPA and TSA were compared to examine the mechanism of differentiation-promoting effects of these drugs. We found that expression of several proteins participating in cell cytoskeleton rearrangement, heat shock response, proteosynthesis and cell signaling was altered in TPA- or TSA-treated cells. We present here the first comparative proteome analysis of v-myb-transformed monoblasts BM2 focused on identification of proteins involved in their terminal differentiation.

Animals↗
PubMed2002

Adapting chromophore-assisted laser inactivation for high throughput functional proteomics.

Recent advances in genomics and proteomics have generated a change in emphasis from hypothesis-based to discovery-based investigations. Genomic and proteomic studies based on differential expression microarrays or comparative proteomics often provide many potential candidates for functionally important roles in normal and diseased cells. High throughput technologies to address protein and gene function in situ are still necessary to exploit these emerging advances in gene and protein discovery in order to validate these identified targets. The pharmaceutical industry is particularly interested in target validation, and has identified it as the critical early step in drug discovery. An especially powerful approach to target validation is a direct protein knockdown strategy called chromophore-assisted laser inactivation (CALI) which is a means of testing the role of specific proteins in particular cellular processes. Recent developments in CALI allow for its high throughput application to address many proteins in tandem. Thus, CALI may have applications for high throughput hypothesis testing, target validation or proteome-wide screening.

Drug Industry↗
PubMed2004

A proteomic analysis of maize chloroplast biogenesis.

Proteomics studies to explore global patterns of protein expression in plant and green algal systems have proliferated within the past few years. Although most of these studies have involved mapping of the proteomes of various organs, tissues, cells, or organelles, comparative proteomics experiments have also led to the identification of proteins that change in abundance in various developmental or physiological contexts. Despite the growing use of proteomics in plant studies, questions of reproducibility have not generally been addressed, nor have quantitative methods been widely used, for example, to identify protein expression classes. In this report, we use the de-etiolation ("greening") of maize (Zea mays) chloroplasts as a model system to explore these questions, and we outline a reproducible protocol to identify changes in the plastid proteome that occur during the greening process using techniques of two-dimensional gel electrophoresis and mass spectrometry. We also evaluate hierarchical and nonhierarchical statistical methods to analyze the patterns of expression of 526 "high-quality," unique spots on the two-dimensional gels. We conclude that Adaptive Resonance Theory 2-a nonhierarchical, neural clustering technique that has not been previously applied to gene expression data-is a powerful technique for discriminating protein expression classes during greening. Our experiments provide a foundation for the use of proteomics in the design of experiments to address fundamental questions in plant physiology and molecular biology.

Chloroplasts↗
PubMed2002

Preferred codons and amino acid couples in hyperthermophiles.

BACKGROUND: Most organisms grow at temperatures from 20 to 50 degrees C but some prokaryotes, including Archaea and Bacteria, are capable of withstanding higher temperatures, from 60 to >100 degrees C. What makes these cells so resistant to heat? Their biomolecules must be sufficiently stable, especially proteins, to work under these extreme conditions, but the bases for thermostability remains elusive. RESULTS: The preferential usage of certain couples of amino acids and codons in thermal adaptation was investigated, by comparative proteome analysis, using 28 complete genomes from 18 mesophiles, 4 thermophiles, and 6 hyperthermophiles. In the hyperthermophiles proteomes, whenever the percent of Glu (E) and Lys (K) Increased, the percent of Gln (Q) and His (H) decreased, so that the E+K/Q+H ratio was > 4,5; in the mesophiles proteomes, it was < 2,5 and in the thermophiles an intermediary value was observed. The E+K/Q+H ratios for chaperonins, potentially thermostable proteins, were higher than their proteome ratios whereas, for DNA ligases, not necessarily thermostable, they followed the proteome ones. Analysis of codon usage revealed that hyperthermophiles preferred AGR codons for Arg in detriment of CGN codons, which were preferred by mesophiles. CONCLUSIONS: The results suggested that the E+K/Q+H ratio may provide a useful mark for distinguishing hyperthermophilic, thermophilic and mesophilic prokaryotes and that the high percent of the amino acid couple E+K, consistently associated to the low percent of the pair Q+H, could contribute to protein thermostability. Second, the preference for AGR codons for Arg was a signature of all hyperthermophilics so far analyzed.

Amino Acids↗
PubMed2006

Alterations in the mitochondrial proteome of adriamycin resistant MCF-7 breast cancer cells.

A 2D-gel based comparative proteomic analysis has been conducted of changes in mitochondrial protein abundances in MCF-7 human breast cancer cells selected for resistance to Adriamycin accompanied by verapamil. We identified and compared 156 unique proteins from 184 spots. Eleven mitochondrial proteins were found with abundances altered more than 2-fold. Transcription was evaluated for two of these, using quantitative RT-PCR. Implications of the changes are considered with respect to drug resistance.

Breast Neoplasms↗
PubMed2005

Proteomic analysis of sera from hepatocellular carcinoma patients after radiofrequency ablation treatment.

Comparative proteomic analysis was used to search for characteristic alterations in the sera of hepatocellular carcinoma (HCC) patients who had undergone curative radiofrequency ablation treatment. Serum samples collected from eight patients before and after treatment were subjected to 2-DE. Eighty-eight protein spots differentially expressed with the treatment were selected by clustering analysis, and the proteins were identified by MS based on MALDI-TOF/TOF analysis and public database searches. The statistical analysis suggested that four proteins decreased after treatment (pro-apolipoprotein, alpha2-HS glycoprotein, apolipoprotein A-IV precursor, and PRO1708/PRO2044, which is the carboxy terminal fragment of albumin) and that seven proteins were increased after treatment, including leucine-rich alpha2-glycoprotein and alpha1-antitrypsin. These data facilitate the identification of differentially expressed proteins that are involved in HCC carcinogenesis and provide candidate biomarkers for the development of diagnostic and therapeutic tools.

Apolipoproteins A↗
PubMed2006

Analysis of potential diagnostic biomarkers in cerebrospinal fluid of idiopathic normal pressure hydrocephalus by proteomics.

BACKGROUND: The pathogenesis of idiopathic normal pressure hydrocephalus (INPH) is unknown, and the syndrome of INPH remains a diagnostic and therapeutic challenge. The present study investigated the disease-specific proteins that aid in the diagnosis and treatment of INPH and thus to study their role in the disease process. METHODS: A comparative proteomic analysis was used for clinical screening of cerebrospinal fluid (CSF) proteins in 15 patients with INPH and compared with 12 normal subjects. Furthermore, enzyme linked immunosorbent assay (ELISA) was performed for comparison with CSF proteins between individual INPH patients and controls. RESULTS: Seven proteins and their isoforms, including leucine-rich alpha-2-glycoprotein (LRG), alpha1-antichymotrypsin, apolipoprotein D, apolipoprotein J, haptoglobin alpha1, serum albumin, and alpha-1-microglobulin/bikunin precursor showed significant changes in CSF of INPH patients compared with controls by proteomic analysis. And significant higher CSF levels of LRG in INPH patients compared with controls were found by ELISA. CONCLUSIONS: These results indicate that there are significant differences in the expression of certain proteins in the CSF of patients with INPH and normal subjects. In particular, the CSF level assay of LRG suggests that LRG is a specific biomarker for INPH and has potential use in the diagnosis and indication for CSF shunting.

Aged↗
PubMed2005

Proteomic profiling unravels insights into the molecular background underlying increased Aphanomyces euteiches-tolerance of Medicago truncatula.

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity.

Abscisic Acid↗
PubMed2006

Numerous and rapid nonstochastic modifications of gene products in newly synthesized Brassica napus allotetraploids.

Polyploidization is a widespread process that results in the merger of two or more genomes in a common nucleus. To investigate modifications of gene expression occurring during allopolyploid formation, the Brassica napus allotetraploid model was chosen. Large-scale analyses of the proteome were conducted on two organs, the stem and root, so that >1600 polypeptides were screened. Comparative proteomics of synthetic B. napus and its homozygous diploid progenitors B. rapa and B. oleracea showed that very few proteins disappeared or appeared in the amphiploids (<1%), but a strikingly high number (25-38%) of polypeptides displayed quantitative nonadditive pattern. Nonstochastic gene expression repatterning was found since 99% of the detected variations were reproducible in four independently created amphiploids. More than 60% of proteins displayed a nonadditive pattern closer to the paternal parent B. rapa. Interspecific hybridization triggered the majority of the deviations (89%), whereas very few variations (approximately 3%) were associated with genome doubling and more significant alterations arose from selfing (approximately 9%). Some nonadditive proteins behaved similarly in both organs, while others exhibited contrasted behavior, showing rapid organ-specific regulation. B. napus formation was therefore correlated with immediate and directed nonadditive changes in gene expression, suggesting that the early steps of allopolyploidization repatterning are controlled by nonstochastic mechanisms.

Biological Evolution↗
PubMed2005

Proteomic analysis on metastasis-associated proteins of human hepatocellular carcinoma tissues.

PURPOSE: A comparative proteomic approach was used to identify and analyze proteins related to metastasis of hepatocellular carcinoma (HCC). METHODS: Proteins extracted from 12 HCC tissue specimens (six with metastases and six without) were separated by two-dimensional gel electrophoresis (2-DE). The protein spots exhibiting statistical alternations between the two groups through computerized image analysis were then identified by mass spectrometry. In addition immunohistochemistry (IHC), Western blotting and RT-PCR were performed to verify the expression of certain candidate proteins. RESULTS: 16 proteins including HSP27, S100A11, CK18 were annotated by mass spectrometry, relevant to chaperone function, cell mobility, cytoskeletal architecture, respectively. Most were previously unconnected with metastasis of HCC. Of these HSP27 was found overexpressed consistently in 2-DE patterns of all metastatic HCC tissues compared with nonmetastatic ones. IHC and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. CONCLUSION: There are various proteins joined together in HCC metastasis. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets unique to the metastatic phenotype of HCC.

Actins↗
PubMed2006

Proteome analysis of human plasma and amniotic fluid by Off-Gel isoelectric focusing followed by nano-LC-MS/MS.

This paper presents a comparative proteomic analysis of human maternal plasma and amniotic fluid (AF) samples from the same patient at term of pregnancy in order to find specific AF proteins as markers of premature rupture of membranes, a complication frequently observed during pregnancy. Maternal plasma and the corresponding AF were immunodepleted in order to remove the six most abundant proteins before the systematic analysis of their protein composition. The protein samples were then fractionated by IEF Off-Gel electrophoresis (OGE), digested and analyzed with nano-LC-MS/MS separation, revealing a total of 73 and 69 proteins identified in maternal plasma and AF samples, respectively. The proteins identified in AF have been compared to those identified in the mother plasma as well as to the reference human plasma protein list reported by Anderson et al. (Mol. Cell. Proteomics 2004, 3, 311-326). This comparison showed that 26 proteins were exclusively present in AF and not in plasma among which 10 have already been described to be placenta or pregnancy specific. As a further validation of the method, plasma proteins fractionated by OGE and analysed by nano-LC-MS/MS have been compared to the Swiss 2-D PAGE reference map by reconstructing a map that matches 2-D gel and OGE experimental data. This representation shows that 36 of 49 reference proteins could be identified in both data sets, and that isoform shifts in pI are well conserved in the OGE data sets.

Amniotic Fluid↗
PubMed2006

[Proteomic approach to the effect of epirubicin on hepatoma cells at subcellular level].

Epirubicin is an antineoplastic agent known as an anthracycline. It acts directly on DNA by blocking its replication and transcription, so apoptosis can be induced for cancer cells. The protein expression of cancer cells will be altered due to the induction of pharmacological action of epirubicin, so it is important to pay attention to the altered profiling of proteins. Here proteomic strategy was applied to the hepatoma cells at subcellular level, comparative proteome analysis of mitochondria and nucleus were conducted between the hepatoma cells administered with epirubicin and the not-administered. Centrifugation was used for the subcellular fractionation, then 2-DE for the separation of proteins, imaging analysis for the diction of expression-altered spots, and MALDI-TOF-MS for the identification of proteins. In total, 15 proteins were found to have altered their expression after the induction of epirubicin, among them, 5 proteins showed up-regulated expression and 10 showed down-regulated expression. These altered proteins are involved in life processes of cells such as energy metabolism, protein biosynthesis, structure of cell skeleton, processing and maturation of mRNA, heat shock of cells and apoptosis.

Carcinoma, Hepatocellular↗
PubMedDate not supplied

Proteomic analysis of Trypanosoma cruzi developmental stages using isotope-coded affinity tag reagents.

Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC-MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC-MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes.

Amino Acid Sequence↗
PubMed2006

Beta-amyloid treatment of two complementary P301L tau-expressing Alzheimer's disease models reveals similar deregulated cellular processes.

Alzheimer's disease (AD) is characterized by Abeta peptide-containing plaques and tau-containing neurofibrillary tangles (NFTs). Both pathologies have been combined by crossing Abeta plaque-forming APP mutant mice with NFT-forming P301L tau mutant mice or by stereotaxically injecting beta-amyloid peptide 1-42 (Abeta42) into brains of P301L tau mutant mice. In cell culture, Abeta42 induces filamentous tau aggregates. To understand which processes are disrupted by Abeta42 in the presence of tau aggregates, we applied comparative proteomics to Abeta42-treated P301L tau-expressing neuroblastoma cells and the amygdala of P301L tau transgenic mice stereotaxically injected with Abeta42. Remarkably, a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. We also identified model-specific effects of Abeta42 treatment such as differences in cell signaling proteins in the cellular model and of cytoskeletal and synapse associated proteins in the amygdala. By Western blotting (WB) and immunohistochemistry (IHC), we were able to show that 72% of the tested candidates were altered in human AD brain with a major emphasis on stress-related unfolded protein responsive candidates. These data highlight these processes as potentially important initiators in the Abeta42-mediated pathogenic cascade in AD and further support the role of unfolded proteins in the course of AD.

Alzheimer Disease↗
PubMedDate not supplied

Global protein shotgun expression profiling of proliferating mcf-7 breast cancer cells.

Protein expression becomes altered in breast epithelium during malignant transformation. Knowledge of these perturbations should provide insight into the molecular basis of breast cancer, as well as reveal possible new therapeutic targets. To this end, we have performed an extensive comparative proteomic survey of global protein expression patterns in proliferating MCF-7 breast cancer cells and normal human mammary epithelial cells using gel-free shotgun tandem mass spectrometry. Pathophysiological alterations associated with the malignant breast cancer phenotype were detected, including differences in the apparent levels of key regulators of the cell cycle, signal transduction, apoptosis, transcriptional regulation, and cell metabolism.

Apoptosis↗
PubMed2004

Characterization of RNase-like major storage protein from the ginseng root by proteomic approach.

The most abundant root proteins of ginseng (Panax ginseng) have been detected and identified by comparative proteome analysis with cultured hairy root of ginseng. Four abundant proteins (28, 26, 21 and 20 kDa) of P. ginseng had isoforms with different pl values on two-dimensional gel electrophoresis (2DE). The results of N-terminal and internal amino acid sequencing, however, showed that all of them originate from a 28 kDa protein, known as ginseng major protein (GMP). The GMP gene was searched for in the expressed sequence tag database of P. ginseng and found to encode a 27.3 kDa protein having 238 amino acid residues. Analysis of the amino acid sequences indicates that GMP exhibits high sequence homology with plant RNases and RNase-like proteins. However, purified GMP had no RNase activity even though it has conserved amino acid residues known to be essential for active sites of RNase. The GMPs present in ginseng main root were not expressed in cultured hairy roots of ginseng. 2DE analysis showed that the amounts of GMPs in main roots change according to seasonal fluctuation. These results suggest that the GMPs are root-specific RNase-like proteins, which function as vegetative storage proteins of ginseng for survival in the natural environment.

Amino Acid Sequence↗