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PubMed2006

Comparative proteome analysis of subcellular fractions from Borrelia burgdorferi by NEPHGE and IPG.

Borrelia burgdorferi, the cause of Lyme disease, produces excessive amounts of membrane lipoproteins such as outer surface protein A (OspA) when grown in vitro, and consequently many low or moderately abundant proteins are underrepresented when cell lysates are examined by 2-DE. We analyzed the B. burgdorferi B31 proteome computationally and by IPG or modified NEPHGE after subcellular fractionation into membrane-associated and soluble proteins. The B. burgdorferi B31 theoretical proteome is comprised of 1623 proteins and has a mean pI of 8.36 and a median pI of 9.03 with 68% of the proteome possessing a pI >/=7.5. Separation of soluble proteins by IPG resulted in 205 individual spots and identification of 78 protein spots by MALDI-TOF MS. Separation by modified NEPHGE routinely resulted in approximately 185 soluble and 160 membrane protein spots with the identification of 88 individual protein spots combined by MALDI-TOF MS. Homologues to GroEL and aminopeptidase I were present in greater amounts in the membrane faction, with enolase at nearly equivalent amounts in the soluble and membrane fractions. Identification of proteins isolated and separated by such methods will enable future determination of proteome changes in membrane and soluble protein fractions as spirochetes adapt to their changing environments.

Bacterial Proteins↗
PubMed2006

Comparative proteome analysis of Staphylococcus aureus biofilm and planktonic cells and correlation with transcriptome profiling.

Pathogenic staphylococci can form biofilms in which they show a higher resistance to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have an altered metabolic activity. Here, 2-D PAGE was used to identify secreted, cell wall-associated and cytoplasmic proteins expressed in Staphylococcus aureus after 8 and 48 h of growth. The proteins were separated at pH ranges of 4-7 or 6-11. The protein patterns revealed significant differences in 427 protein spots; from these, 258 non-redundant proteins were identified using ESI-MS/MS. Biofilm cells expressed higher levels of proteins associated with cell attachment and peptidoglycan synthesis, and in particular fibrinogen-binding proteins. Enzymes involved in pyruvate and formate metabolism were upregulated. Furthermore, biofilm cells expressed more staphylococcal accessory regulator A protein (SarA), which corroborates the positive effect of SarA on the expression of the intercellular adhesion operon ica and biofilm growth. In contrast, proteins, such as proteases and particularly immunodominant antigen A (IsaA) and staphylococcal secretory antigen (SsaA), were found in lower amounts. The RNA expression profiling largely supports the proteomic data. The results were mapped onto KEGG pathways.

Bacterial Proteins↗
PubMed2005

[Comparative proteomics research on removing of large invasive plasmid pINV of Shigella flexneri 2a strain 2457T].

S. flexneri 2a strain 2457T and its derivative without large invasive plasmid pINV-2457T were cultured to middle logarithm phase. Whole cellular protein extracts of the two strains were examined by two dimensional (2D) electrophoresis using immobilized pH gradient (IPG) technology. After in-gel protein digestion, the different-expressed spots were detected by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS). All the peptide mass fingerprints (PMFs) were searched by the program Mascot developed by Matrix Science Ltd. For identifying proteins, databases of S. flexneri 2a 2457T was used. Ten proteins with changed expression level were found. Results indicate that expression levels of several enzymes involved in nucleic acid metabolism have risen, and expression increase of deoxycytidine deaminase, purine nucleoside phosphorylase, and uridine nucleoside phosphorylase might lead to increase of uridine and uridine nucleoside synthesization.

Bacterial Proteins↗
PubMed2004

Comparative proteome analysis of Hansenula polymorpha DL1 and A16.

Proteomic responses of methylotrophic yeasts (Hansenula polymorpha DL1 and A16) to growth medium tuning by carbon source shift (glycerol-->methanol) were monitored and analyzed by two-dimensional gel electrophoresis. Through comparative analyses of two-dimensional gels, intracellular yeast proteins with complex expression patterns were systematically sorted into: (1) proteins that are commonly expressed with comparable high abundance in both strains; (2) strain-specific proteins that are expressed at high level only in a particular strain; (3) strain-specific and methanol-induced proteins that are expressed only in the presence of methanol; and (4) strain-specific and constitutively-expressed proteins that are expressed consistently irrespective of carbon source shift without extreme change in expression level. Among the DL1-specific proteins belonging to group four, the four proteins showing the highest expression levels in the course of the fermentation process were identified as: glucose-6-phosphate dehydrogenase, isocitrate lyase, succinyl-CoA synthetase, and glycerol-3-phosphate dehydrogenase. From these results, it is suggested that DL1 has distinct metabolic characteristics including enhanced metabolic activities both in glycerol uptake and the glyoxylate bypass cycle, as compared to A16. This is likely to explain why the DL1 strain shows a significantly higher rate of glycerol and methanol consumption during the fermentation process. Our systematic approach to the analysis of proteomic responses and the detailed analysis results reported here will be useful to better understand the global physiology of H. polymorpha, as proteome databases for various methylotrophic yeasts are established.

Carbon↗
PubMed2006

Potential biomarkers for ischemic heart damage identified in mitochondrial proteins by comparative proteomics.

We used proteomics to detect regional differences in protein expression levels from mitochondrial fractions of control, ischemia-reperfusion (IR), and ischemic preconditioned (IPC) rabbit hearts. Using 2-DE, we identified 25 mitochondrial proteins that were differentially expressed in the IR heart compared with the control and IPC hearts. For three of the spots, the expression patterns were confirmed by Western blotting analysis. These proteins included 3-hydroxybutyrate dehydrogenase, prohibitin, 2-oxoglutarate dehydrogenase, adenosine triphosphate synthases, the reduced form of nicotinamide adenine dinucleotide (NADH) oxidoreductase, translation elongation factor, actin alpha, malate dehydrogenase, NADH dehydrogenase, pyruvate dehydrogenase and the voltage-dependent anion channel. Interestingly, most of these proteins are associated with the mitochondrial respiratory chain and energy metabolism. The successful use of multiple techniques, including 2-DE, MALDI-TOF-MS and Western blotting analysis demonstrates that proteomic analysis provides appropriate means for identifying cardiac markers for detection of ischemia-induced cardiac injury.

Animals↗
PubMed2006

Comparative proteomic analysis of human leukemic cells with and without inducible expression of leukemogenic AML1-ETO protein.

AML1-ETO is a leukemogenic fusion protein generated by chromosomal translocation t(8; 21) (q22; q22), one of the most frequent chromosomal abnormalities in acute myeloid leukemia. The fusion protein has been shown to present dichotomous functions on leukemic cells: growth arrest versus differentiation block. However, their precise molecular mechanisms are not completely clear. In this work, we try to explore potential AML1-ETO-targeted proteins through comparing two-dimensional electrophoresis (2DE)-based global protein expression profiles of leukemic U937 cells with and without inducible expression of AML1-ETO. As a result, we identified 14 unique proteins deregulated in AML1-ETO-carrying leukemic cells, including 3 up-regulated such as hairy and enhancer of split 5 (HES5) and 11 down-regulated such as MAT1 (menage a trois-1) and mitogen-activated protein kinase organizer 1 (MORG1). These proteins were widely involved in stem cell maintenance, cell cycle, signal transduction and transcription. The further investigation on their roles in leukemic cells will uncover new clues to understanding leukemogenic effects of AML1-ETO fusion protein.

Cell Line, Tumor↗
PubMed2006

Identification of genetic markers to distinguish the virulent and avirulent subspecies of Pantoea stewartii by comparative proteomics and genetic analysis.

Pantoea stewartii subsp. stewartii (Pnss), the causal agent of Stewart's bacterial wilt and leaf blight of maize and sweet corn, is one of the quarantine pathogens in many countries and regions. In contrast, P. stewartii subsp. indologenes (Pnsi), the closely related subspecies of Pnss, is avirulent on these plants. In this study, the protein expression profiles of these two subspecies were compared using two-dimensional gel electrophoresis analysis. Twenty-one unique protein spots consistently detected in Pnss but not in Pnsi were analyzed by mass spectrometry. Some of these Pnss-specific proteins are known to be essential for virulence and survival in host, such as FoxR and HrcJ, which are the key components of iron uptake and Type III secretion systems, respectively. For further genetic analysis, six Pnss-specific proteins were characterized by peptide sequencing. Southern and Northern blot analyses revealed that the differences in protein expression profiles of the two subspecies were either due to the discrepancy at genome level or because of the variations in transcriptional expression. The results provide novel genetic markers to distinguish the two closely related subspecies and may also serve as useful clues for investigation of the genetic basis accounting for their sharp difference in virulence.

Amino Acid Sequence↗
PubMed2005

A comparative proteomic analysis of the rat brain during rebound hyperphagia induced by space-restriction.

Although neurochemical changes have been reported in the brain in animal models of binge eating, biochemical changes of specific proteins in the brain are unknown. Our aim was to elucidate brain proteins altered in rats during enhanced rebound hyperphargia. Rats were deprived of food for 22 h/day for 6 days, then allowed free access to food for 24 h in normal cages (rebound hyperphargia) or in space-restricted cages (enhanced rebound hyperphargia). Proteins extracted from the rat brain were separated by two-dimensional gel electrophoresis, and compared with those from control rats freely fed for 7 days in normal cages. Proteins expressed differently from controls were identified by N-terminal amino acid sequencing and mass fingerprinting using a MALDI-TOF mass spectrometer. Among proteins in the corpus striatum, frontal lobe, hippocampus and thalamus/hypothalamus, ubiquitin C-terminal hydrolase L1 and peroxiredoxin 2 decreased in the hippocampus and phosphatidylethanolamine-binding protein increased in the thalamus/hypothalamus of rats with the enhanced rebound hyperphargia induced by space-restriction. In this study, we first demonstrated that three brain proteins changed in rats during enhanced rebound hyperphagia. These proteins might have pathophysiologic relevance to binge eating.

Animals↗
PubMed2002

Global internal standard technology for comparative proteomics.

The work described in this paper tests the efficacy of a global isotope labeling (global internal standard technology, GIST) strategy for quantification in proteomics. Using GIST, overexpression of beta-galactosidase in Escherichia coli was identified and quantified. The GIST protocol involved tryptic digestion of proteins from control and experimental samples followed by differential isotopic labeling of the resulting tryptic peptides, mixing the differentially labeled control and experimental digests, fractionation of the peptide mixture by reversed-phase chromatography, and isotope ratio analysis by mass spectrometry. N-Acetoxysuccinimide and N-acetoxy-[2H3]succinimide were used to differentially derivatize primary amino groups in peptides from experimental and control samples, respectively. The relative concentration of isotopically labeled peptides was determined by isotope ratio analysis with both matrix-assisted laser desorption ionization mass spectrometry and tandem mass spectrometry (MS-MS). Peptide masses and sequences obtained by MS-MS were used to identify proteins. MS-MS was found to be uniquely suited for isobaric peptide quantification.

Amino Acid Sequence↗
PubMed2002

Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders.

More than 70 different point mutations in human mitochondrial tRNA genes are correlated with severe disorders, including fatal cardiopathies, encephalopathies, myopathies, and others. So far, investigation of the molecular impact(s) of mutations has focused on the affected tRNA itself by seeking structural and/or functional perturbations capable of interfering with synthesis of the 13 mitochondrion-encoded subunits of respiratory chain complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Analysis of several hundred mitochondrial proteins from sibling cybrid cell lines by two-dimensional electrophoresis, an approach that takes into account all regulatory steps of mitochondrial and nuclear gene expression, indeed reveals a number of up- and downregulated proteins when healthy and single-point-mutation-carrying mitochondria representative of either MELAS or MERRF syndrome were compared. Assignment by mass spectrometry of the two proteins which exhibit obvious large quantitative decreases in the levels of both pathologic mitochondria identified nuclear-encoded subunits of cytochrome c oxidase, a respiratory chain complex. This clearly shows a linkage between the effects of mutations in mitochondrial tRNA genes and the steady-state level of nuclear-encoded proteins in mitochondria. It opens new routes toward a large-scale exploration of potential proteic partners involved in the genotype-phenotype correlation of mitochondrial disorders.

Amino Acid Sequence↗
PubMed2006

Comparative proteomics analysis of Barrett metaplasia and esophageal adenocarcinoma using two-dimensional liquid mass mapping.

Esophageal adenocarcinoma, currently the seventh leading cause of cancer-related death, has been associated with the presence of Barrett metaplasia. The malignant potential of Barrett metaplasia is evidenced by ultimate progression of this condition to invasive adenocarcinoma. We utilized liquid phase separation of proteins with chromatofocusing in the first dimension and nonporous reverse phase HPLC in the second dimension followed by ESI-TOF mass spectrometry to identify proteins differentially expressed in six Barrett metaplasia samples as compared with six esophageal adenocarcinoma samples; all six Barrett samples were obtained from the identical six patients from whom we obtained the esophageal adenocarcinoma tissue. Approximately 300 protein bands were detected by mass mappings, and 38 differentially expressed proteins were identified by microLC-MS/MS. The false positive rates of the peptide identifications were evaluated by reversed database searching. Among the proteins that were identified, Rho GDP dissociation inhibitor 2, alpha-enolase, Lamin A/C, and nucleoside-diphosphate kinase A were demonstrated to be up-regulated in both mRNA and protein expression in esophageal adenocarcinomas relative to Barrett metaplasia. Candidate proteins were examined at the mRNA level using high density oligonucleotide microarrays. The cellular expression patterns were verified in both esophageal adenocarcinomas and in Barrett metaplasia by immunohistochemistry. These differentially expressed proteins may have utility as useful candidate markers of esophageal adenocarcinoma.

Adenocarcinoma↗
PubMed2006

Comparative proteomics of rat liver and Morris hepatoma 7777 plasma membranes.

Plasma membranes from normal rat liver and hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized by use of different reagents. After selective solubilization, proteins were identified by nano-HPLC-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Using simple software, the patterns of proteins identified in membrane solubilizates from liver and hepatoma were compared. Proteins identified in Morris hepatoma 7777 and not in the corresponding membrane solubilizate from liver, mostly members of the annexin and heat shock protein families, are discussed as potential candidate markers for hepatocellular carcinomas.

Amino Acid Sequence↗
PubMed2004

Comparative proteomics of human endothelial cell caveolae and rafts using two-dimensional gel electrophoresis and mass spectrometry.

The human endothelial cell plasma membrane harbors two subdomains of similar lipid composition, caveolae and rafts, both crucially involved in various essential cellular processes like transcytosis, signal transduction and cholesterol homeostasis. Caveolin-enriched membranes, isolated by either cationic silica or buoyant density methods, were explored by comparing large series of two-dimensional (2-D) maps and subsequent identification of over 100 protein spots by matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting. Improved representation and identification of membrane proteins and valuable information on various post-translational modifications was achieved by the presented optimized procedures for solubilization, destaining and database searching/computing. Whereas the cationic silica purification yielded predominantly known endoplasmic reticulum residents, the cold-detergent method yielded a large number of known caveolae residents, including caveolin-1. Thus, a large part of this subproteome was established, including known (trans-)membrane, signal transduction and glycosyl phosphatidylinositol (GPI)-anchored proteins. Several predicted proteins from the human genome were isolated for the first time from biological samples, including SGRP58, SLP-2, C8ORF2, and XRP-2. These findings and various optimized procedures can serve as a reference to study the differential composition of endothelial cell caveolae and rafts, known to be involved in pathologies like cancer and cardiovascular disease.

Blood Proteins↗
PubMed2003

Comparative proteomic profiling of murine skin.

Mammalian skin is regularly exposed to different environmental stresses, each of which results in specific compensatory changes in protein expression that can be assessed by proteomic analysis. We have established a reference proteome map of BALB/c murine skin allowing the resolution of greater than 500 protein spots in a single two-dimensional polyacrylamide gel. Forty-four protein spots, corresponding to 28 different cutaneous proteins, were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. Twenty-five proteins were expressed at higher levels in the epidermis, whereas only nine were found predominantly in the subepidermal tissues. A subset of protein spots exhibited strain-specific expression. Proteins of diverse function were identified, including those involved in stress response, apoptosis, growth inhibition, the maintenance of structural integrity, translational control, energy metabolism, calcium binding, cholesterol transport, and the scavenging of free radicals. Prohibitin expression was detected cutaneously, with more abundant protein and mRNA levels in the epidermis. Five molecular chaperones including protein di-sulfide isomerase, 78 kDa glucose-regulated protein precursor, heat shock protein 60 (HSP60), HSP70, and HSP27 were also identified. Of these, HSP27 expression was confined mainly to the epidermis, and expression of protein disulfide isomerase was found primarily in the subepidermal tissues. Proteomic analysis of skin following heat or cold shock resulted in increased levels of HSP27, HSP60, and HSP70 suggesting involvement of these chaperones in the cutaneous response mechanism to temperature stress. These data establish numerous reference markers within the proteome map of murine skin and provide an important framework for future efforts aimed at characterization of the epidermal and subepidermal responses to environmental changes.

Abdomen↗
PubMed2004

Comparative proteomic analysis of all-trans-retinoic acid treatment reveals systematic posttranscriptional control mechanisms in acute promyelocytic leukemia.

All-trans-retinoic acid (ATRA) induces growth inhibition, differentiation, and apoptosis in cancer cells, including acute promyelocytic leukemia (APL). In APL, expression of promyelocytic leukemia protein retinoic acid receptor-alpha (PML-RARalpha) fusion protein, owing to the t(15; 17) reciprocal translocation, leads to a block in the promyelocytic stage of differentiation. Here, we studied molecular mechanisms involved in ATRA-induced growth inhibition and myeloid cell differentiation in APL. By employing comprehensive high-throughput proteomic methods of 2-dimensional (2-D) gel electrophoresis and amino acid-coded mass tagging coupled with electrospray ionization (ESI) mass spectrometry, we systematically identified a total of 59 differentially expressed proteins that were consistently modulated in response to ATRA treatment. The data revealed significant down-regulation of eukaryotic initiation and elongation factors, initiation factor 2 (IF2), eukaryotic initiation factor 4AI (eIF4AI), eIF4G, eIF5, eIF6, eukaryotic elongation factor 1A-1 (eEF1A-1), EF-1-delta, eEF1gamma, 14-3-3epsilon, and 14-3-3zeta/delta (P <.05). The translational inhibitor DAP5/p97/NAT1 (death-associated protein 5) and PML isoform-1 were found to be up-regulated (P <.05). Additionally, the down-regulation of heterogeneous nuclear ribonucleoproteins (hnRNPs) C1/C2, UP2, K, and F; small nuclear RNPs (snRNPs) D3 and E; nucleoprotein tumor potentiating region (TPR); and protein phosphatase 2A (PP2A) were found (P <.05); these were found to function in pre-mRNA processing, splicing, and export events. Importantly, these proteomic findings were validated by Western blot analysis. Our data in comparison with previous cDNA microarray studies and our reverse transcription-polymerase chain reaction (RT-PCR) experiments demonstrate that broad networks of posttranscriptional suppressive pathways are activated during ATRA-induced growth inhibition processes in APL.

Antineoplastic Agents↗
PubMed2002

Comparative proteome analysis of human temporal cortex lobes by two-dimensional electrophoresis and identification of selected common proteins.

Human brain proteins were isolated from left and right temporal cortex lobes at the age of 73, 23, 84 years and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient strip in the first dimension and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over 800 polypeptide spots were resolved with a silver-staining protocol by computerized 2-D gel analsis. Seven of the polypeptide spots were evidently distinguishable between human left and right temporal lobes. Four of the polypeptide spots were larger and three were smaller in human right temporal lobe. One of these three protein spots that have descendent expression in human right temporal lobe was identified as carbonyl reductase (NADPH) 1 by MALDI-TOF MS. Thirty-three common spots were identified by ESI-MS/MALDI-TOF MS/Edman sequencing and a protein database search. These identified proteins include some important enzymes and regulating proteins.

Amino Acid Sequence↗
PubMed2003

[Comparative proteomics research of apoptosis initiation induced by homoharringtonine in HL-60 cells].

OBJECTIVE: To study the related proteins of apoptosis initiation induced by homoharringtonine (HHT) in HL-60 cells. METHODS: After establishment of an apoptosis initiation model induced by HHT in HL-60 cells, proteins of untreated and HHT treated HL-60 cells were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2-DE) maps of the extracted proteins were established by using the immobilized pH gradient (IPG) two-dimensional electrophoresis respectively. The alteration protein spots were identified with assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. RESULTS: Proteomics analysis showed that proteins including MHC class I antigen, calbindin D-28K, chloride channel protein 6, oncoprotein 18, zinc finger protein Helios and apoptosis inhibitor like protein 2 were involved in apoptosis initiation induced by HHT. CONCLUSION: The present study might conduce to the researches of HL-60 cells carcinogenesis and pave the way to exploit drug precursor related to HHT and initiation of apoptosis in HL-60 cells.

Antineoplastic Agents, Phytogenic↗
PubMed2003

Comparative proteome analysis of native differentiated and cultured dedifferentiated human RPE cells.

PURPOSE: Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells. METHODS: Differentiated native human RPE cells and monolayers of dedifferentiated cultured primary human RPE cells were processed for two-dimensional (2-D) electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3-10) and electrophoresis on 9% to 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver-stained gel spots were excised, digested in situ, and analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy (MS). The resultant peptide mass fingerprints were searched against the public domain NCBInr, MSDB, and EnsemblC databases to identify the respective proteins. RESULTS: One hundred seventy nine protein spots were analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells, including RPE65, cellular retinaldehyde-binding protein (CRALBP), and cellular retinol-binding protein (CRBP), were absent in dedifferentiated cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including cathepsin D and clathrin were still present. Dedifferentiated RPE cells displayed a strong shift toward increased expression of proteins associated with cell shape, cell adhesion, and stress fiber formation, including cytokeratin 19, gelsolin, and tropomyosins, and also acquired increased expression of factors involved in translation and tumorigenic signal transduction such as annexin I and translation initiation factor (eIF)-5A. CONCLUSIONS: Dedifferentiation of human RPE cells in vitro results in downregulation of proteins associated with highly specialized functions of the RPE and induces the differential expression of proteins related to cytoskeleton organization, cell shape, cell migration, and mediation of proliferative signal transduction. These in vitro data suggest that the dedifferentiated status of RPE cells per se may initiate PVR. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation.

Cell Differentiation↗