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PubMed2004

Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis.

Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro- and extraintestinal infections in humans. Using a combination of comparative proteomics and TnphoA mutagenesis, we have identified five proteins that may contribute to E. tarda PPD130/91 pathogenesis. Lowered protein secretion, impaired autoaggregation and the absence of six proteins were observed only in three highly attenuated mutants when cultured in Dulbecco's modified eagle medium (DMEM). Five out of six proteins could be identified by their mass spectra. Three proteins were identified as putative effector proteins (EseB, EseC and EseD) that are homologous to SseB, SseC and SseD of a type III secretion system (TTSS) in Salmonella species. The other two were EvpA and EvpC, homologous to Eip20 and Eip18 in Edwardsiella ictaluri. The complete sequencing and homology studies of evpA-H indicate that similar gene clusters are widely distributed in other pathogens such as Escherichia, Salmonella, Vibrio and Yersinia species with unknown functions. Insertional inactivation and deletion of evpB or evpC led to lower replication rates in gourami phagocytes, and reduced protein secretion and virulence in blue gourami. Complementation of these deletion mutants showed partial recovery in the above three phenotypes, indicating that these genes are vital for E. tarda pathogenesis. The transport of the EvpC protein may not use the TTSS in E. tarda. The expression of EvpA and EvpC as well as EseB, EseC and EseD was temperature dependent (suppressed at 37 degrees C), and disruption of esrB affected their expression. The present study identifies two possible secretion systems (TTSS and Evp) that are vital for E. tarda pathogenesis.

Bacterial Adhesion↗
PubMed2007

Identification of proteomic changes during differentiation of adult mouse subventricular zone progenitor cells.

The use of neural precursor cells (NPCs) represents a promising repair strategy for many neurological disorders. However, the molecular events and biological features that control NPC proliferation and their differentiation into neurons, astrocytes, and oligodendrocytes are unclear. In the present study, we used a comparative proteomics approach to identify proteins that were differentially regulated in NPCs after short-term differentiation. We also used a subcellular fractionation technique for enrichment of nuclei and other dense organelles to identify proteins that were not readily detected in whole cell extracts. In total, 115 distinct proteins underwent expression changes during NPC differentiation. Forty one of these were only identified following subcellular fractionation. These included transcription factors, RNA-processing factors, cell cycle proteins, and proteins that translocate between the nucleus and cytoplasm. Biological network analysis showed that the differentiation of NPCs was associated with significant changes in cell cycle and protein synthesis machinery. Further characterization of these proteins could provide greater insight into the mechanisms involved in regulation of neurogenesis in the adult central nervous system (CNS) and potentially identify points of therapeutic intervention.

Adult Stem Cells↗
PubMed2002

Organellar proteomics: the prizes and pitfalls of opening the nuclear envelope.

Proteomic studies have the potential to comprehensively define the composition of organelles but are limited by the organellar cross-contamination that arises during subcellular fractionation. Comparative proteomics of organellar subfractions can mitigate these problems, as demonstrated by a recent study involving the nuclear envelope.

Animals↗
PubMed2004

Improving large-scale proteomics by clustering of mass spectrometry data.

Tandem mass spectrometry (MS/MS), coupled with liquid chromatography (LC), is a powerful tool for the analysis and comparison of complex protein and peptide mixtures. However, the extremely large amounts of data that result from the process are very complex and difficult to analyze. We show how the clustering of similar spectra from multiple LC-MS/MS runs can help in data management and improve the analysis of complex peptide mixtures. The major effect of spectrum clustering is the reduction of the huge amounts of data to a manageable size. As a result, analysis time is shorter and more data can be stored for further analysis. Furthermore, spectrum quality improvement allows the identification of more peptides with greater confidence, the comparison of complex peptide mixtures is facilitated, and the entire proteomics project is presented in concise form. Pep-Miner is an advanced software tool that implements these clustering-based applications. It proved useful in several comparative proteomics projects involving lung cancer cells and various other cell types. In one of these projects, Pep-Miner reduced 517 000 spectra to 20 900 clusters and identified 2518 peptides derived from 830 proteins. Clustering and identification lasted less than two hours on an IBM Thinkpad T23 computer (laptop). Pep-Miner's unique properties make it a very useful tool for large-scale shotgun proteomics projects.

Algorithms↗
PubMed2006

Neuroproteomics of the synapse and drug addiction.

Proteomic analyses of brain tissues are becoming an integral component of neuroscientific research. In particular, the essential role of the synapse in neurotransmission and plasticity has brought about extensive efforts to identify its protein constituents. Recent studies have used a combination of subcellular fractionation and proteomic techniques to identify proteins associated with different components of the synapse. Thus, a coherent map of the synapse proteome is rapidly emerging, and a timely review of these data is warranted. In the first part of this review, neuroproteomic techniques that have been used to analyze the synapse proteome are described. We then summarize the results from several recent proteomic analyses of mammalian synapses and discuss the similarities and differences in their profiling of synaptic proteins. Important advances in this field of research include the use of proteomics to analyze synaptic function and drug effects on synaptic proteins. This article presents an overview of proteomic analyses of the phosphorylation states of synaptic proteins and recent applications of neuroproteomic techniques to the study of drug addiction. Finally, we discuss the challenges in comparing proteomic studies of drug addiction and the future directions of this field in furthering our understanding of the molecular mechanisms underlying synaptic function and drug addiction.

Animals↗
PubMed2006

Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.

Cell Line↗
PubMed2004

Genome and proteome analysis of Chlamydia.

It has been difficult to study the molecular biology of the obligate intracellular bacterium Chlamydia due to lack of genetic transformation systems. Therefore, genome sequencing has greatly expanded the information concerning the biology of these pathogens. Comparing the genomes of seven sequenced Chlamydia genomes has provided information of the common gene content and gene variation. In addition, the genome sequences have enabled global investigation of both transcript and protein content during the developmental cycle of chlamydiae. During this cycle Chlamydia alternates between an infectious extracellular form and an intracellular dividing form surrounded by a phagosome membrane termed the chlamydial inclusion. Proteins secreted from the chlamydial inclusion into the host cell may interact with host cell proteins and modify the host cell's response to infection. However, identification of such proteins has been difficult because the host cell cytoplasm of Chlamydia infected cells cannot be purified. This problem has been circumvented by comparative proteomics.

Bacterial Physiological Phenomena↗
PubMed2005

Proteomic but not enzyme-linked immunosorbent assay technology detects amniotic fluid monomeric calgranulins from their complexed calprotectin form.

Four proteomic biomarkers (human neutrophil peptide 1 [HNP1], HNP2 [defensins], calgranulin C [Cal-C], and Cal-A) characterize the fingerprint of intra-amniotic inflammation (IAI). We compared proteomic technology using surfaced-enhanced laser desorption-ionization-time of flight (SELDI-TOF) mass spectrometry to enzyme-linked immunosorbent assay (ELISA) for detection of these biomarkers. Amniocentesis was performed on 48 women enrolled in two groups: those with intact membranes (n = 27; gestational age [GA], 26.0 +/- 0.8 weeks) and those with preterm premature rupture of the membranes (PPROM; n = 21; GA, 28.4 +/- 0.9 weeks). Paired abdominal amniotic fluids (aAFs)-vaginal AFs (vAFs) were analyzed in PPROM women. Quantitative aspects of HNP1-3, Cal-C, Cal-A, and calprotectin (a complex of Cal-A with Cal-B) were assessed by ELISA. SELDI-TOF mass spectrometry tracings from 16/48 (33.3%) aAFs and 13/17 (88.2%) vAFs were consistent with IAI (three or four biomarkers present). IAI (by SELDI-TOF mass spectrometry) was associated with increased HNP1-3 and Cal-C measured by ELISA. However, immunoassays detected Cal-A in only 4 of the AFs even though its specific SELDI-TOF mass spectrometry peak was identified in 19/48 AFs. Calprotectin immunoreactivity was decreased in AFs retrieved from women with IAI (P = 0.01). In conclusion, IAI is associated with increased HNP1-3 levels. In the absence of isoform-specific ELISAs, mass spectrometry remains the only way to discriminate the HNP biomarker isoforms. Monomeric Cal-A is not reliably estimated by specific ELISA as it binds to Cal-B to form the calprotectin complex. Cal-C was reliably measured by SELDI-TOF mass spectrometry or specific ELISA.

Amniotic Fluid↗
PubMed2005

Proteomic identification of differentially expressed proteins in the aging murine olfactory system and transcriptional analysis of the associated genes.

Decline in olfactory ability has been associated with aging as well as neurodegenerative disorders. The aim of this study was to gain fundamental insight into molecular events associated with the aging olfactory system. We report a comparative proteomic analysis of the olfactory epithelium (OE) and olfactory bulb (OB) of old (80-week old) and young (6-week old) mice with further analysis of age-related differences in differentially expressed proteins at the mRNA level using real-time RT-PCR. Nine proteins in the OE and 20 in the OB were differentially expressed in old and young mice; of these, aldolase 1, peptidyl prolyl isomerase A, mitochondrial aconitase 2, mitochondrial aldehyde dehydrogenase 2 and albumin 1 were identified in the OE; and ATP synthase isoform 1, enolase 1, ferritin heavy chain, malate dehydrogenase 1, tropomyosin alpha 3 chain and dynamin 1 were identified in the OB. At the transcriptional level, aconitase 2 in the OE and ferritin heavy chain 1 in the OB were differentially expressed with aging, in concordance with the proteomic data. Our results demonstrate an altered proteomic profile of the aged murine olfactory system. The identified proteins fall into three broadly defined functional categories: (i) metabolism, (ii) transport/motility and (iii) stress response. Our transcriptional analysis provides insight into possible mechanisms by which protein expression may be regulated in the OE and OB. The results are discussed in relation to the decrement in olfactory sensitivity with aging.

Age Factors↗
PubMed2004

Quantitative cancer proteomics: stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research.

Microarrays have been the primary means for large-scale analyses of genes implicated in cancer progression. However, more recently a need has been recognized for investigating cancer development directly at the protein level. In this report, we have applied a comparative proteomic technique to the study of metastatic prostate cancer. This technology, termed stable isotope labeling with amino acids in cell culture (SILAC), has recently gained popularity for its ability to compare the expression levels of hundreds of proteins in a single experiment. SILAC makes use of (12)C- and (13)C-labeled amino acids added to the growth media of separately cultured cell lines, giving rise to cells containing either "light" or "heavy" proteins, respectively. Upon mixing lysates collected from these cells, proteins can be identified by tandem mass spectrometry. The incorporation of stable isotopes also allows for a quantitative comparison between the two samples. Using this method, we compared the expression levels for more than 440 proteins in the microsomal fractions of prostate cancer cells with varying metastatic potential. Of these, 60 were found elevated greater than 3-fold in the highly metastatic cells, whereas 22 were reduced by equivalent amounts. Western blotting provided further confirmation of the mass spectrometry-based quantification. Our results demonstrate the applicability of this novel approach toward the study of cancer progression using defined cell lines.

Actinin↗
PubMed2003

Potential for proteomic profiling of Helicobacter pylori and other Helicobacter spp. using a ProteinChip array.

The Helicobacter genus is associated with a wide spectrum of pathologies in the gastrointestinal tract. However, in contrast to Helicobacter pylori, few data are available regarding proteomic characteristics of enterohepatic helicobacters. Proteomic analysis of this genus has predominantly utilised two-dimensional gel electrophoresis methodology. In the present study we applied an innovative technique using ProteinChip arrays coupled with surface-enhanced laser desorption/ionisation time of flight mass spectroscopy to accurately assess the M(r) of proteins for comparative proteomic profiling. We analysed binding of outer membrane fractions to a weak cation exchange array for strains of H. pylori from culture collections and compared these profiles to fresh clinical isolates. In addition, we analysed profiles from Helicobacter pullorum, Helicobacter bilis and 'Helicobacter sp. flexispira'. The system proved rapid, accurate and reproducible. Distinct specific profiles for all the strains studied were identified. However, strains from culture collections that have undergone numerous subcultures had almost identical profiles. In contrast, profiles from fresh clinical isolates were markedly different. Moreover, certain features of the profiles from the enterohepatic species were conserved.

Animals↗
PubMedDate not supplied

Quantitative proteomic analysis of the secretory proteins from rat adipose cells using a 2D liquid chromatography-MS/MS approach.

We have developed two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) and 18O proteolytic labeling strategies to identify and compare levels of secretory proteins with low abundance in the conditioned medium of rat adipose cells without or with insulin stimulation. Culture medium was concentrated and secreted proteins were separated on a RP-HPLC followed by LC-MS/MS analysis. For 18O proteolytic labeling, 16O- to 18O-exchange in the digested peptides from eight individual fractions was carried out in parallel in H2(16)O and H(2)18O with immobilized trypsin, and the ratios of isotopically distinct peptides were measured by mass spectrometry. A total of 84 proteins was identified as secreted adipokines. This large number of secretory proteins comprise multiple functional categories. Comparative proteomics of 18O proteolytic labeling allows the detection of different levels of many secreted proteins as exemplified here by the difference between basal and insulin treatment of adipose cells. Taken together, our proteomic approach is able to identify and quantify the comprehensive secretory proteome of adipose cells. Thus, our data support the endocrine role of adipose cells in pathophysiological states through the secretion of signaling molecules.

Adipocytes↗
PubMed2004

A proteome analysis of the arsenite response in cultured lung cells: evidence for in vitro oxidative stress-induced apoptosis.

Arsenite is well documented as a chemotherapeutic agent capable of inducing cell death. However, the cellular response at the molecular level has not been studied extensively. In the present study, we provide for the first time a proteomic analysis of rat LECs (lung epithelial cells) treated with arsenite, with the aim of identifying defence proteins, probably expressed to protect the cells during the course of arsenic-induced apoptosis. Comparative proteome analysis was conducted on LECs and LECs treated with 40 microM arsenite to identify global changes in their protein expression profiles. Over 1000 protein spots were separated by two-dimensional electrophoresis and visualized by silver staining. Seven proteins changed expression levels significantly and were identified by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry and database searching. The proteins up-regulated were mostly HSPs (heat-shock proteins) and antioxidative stress proteins, including HSP70, aldose reductase, haem oxygenase-1, HSP27, ferritin light chain and alphaB-crystallin. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was down-regulated. Pretreatment with the thiol antioxidants glutathione or N-acetylcysteine before arsenite insult effectively abrogated the induction of these defence proteins and sustained cell viability, whereas antioxidants were protective only at earlier time points if they were added to cells after arsenite. Taken together, our results demonstrate that high levels of arsenite cause oxidative stress-induced apoptosis.

Acetylcysteine↗
PubMed2026

Widespread marine and freshwater distributions of active sulfoquinovose-degrading bacteria.

Sulfoquinovose (SQ), a sulfonated sugar produced on a gigaton scale each year, contributes to global sulfur cycling, yet the microbes and pathways mediating its turnover in the environment have been inferred largely from genomic potential rather than direct activity. Here, we coupled incubations of environmental samples with 13C-labeled SQ to DNA-stable isotope probing to identify active SQ carbon assimilators across estuary, mangrove, and lake ecosystems. In estuarine communities, Vibrio and Cognatishimia incorporated SQ-derived 13C; Novosphingobium dominated in the mangrove, and Agrobacterium in the lake. Pure-culture experiments, coupled with comparative proteomics and gene knockout validation, demonstrated that Vibrio strains degrade SQ via modified sulfoglycolytic Embden-Meyerhof-Parnas and Entner-Doudoroff pathways to produce the environmentally significant organosulfur 2,3-dihydroxypropanesulfonate. Comparative genomic analyses suggested that closely related genome representatives of Novosphingobium, Cognatishimia, and Agrobacterium encode the sulfolytic SQ monooxygenase pathway. A global survey of aquatic microbial genomes indicated that over 9% harbor SQ degradation clusters, supporting a widespread distribution of bacterial SQ catabolic potential in aquatic environments.

Fresh Water↗
PubMed2026

Serum Proteomic Profiling Implicates a Dysregulated Neurohormonal-Inflammatory Axis in Post-Fontan Sinus Tachycardia.

BACKGROUND: Postoperative sinus tachycardia is a poorly understood complication following the Fontan procedure. The molecular signaling cascades triggering acute tachycardia remain uncharacterized, limiting therapeutic innovation. Here, we present a retrospective study leveraging serum proteomics and machine learning to identify the molecular drivers of postoperative Fontan sinus tachycardia. METHODS: We integrated a clinically relevant ovine Fontan model with continuous telemetric heart rate monitoring and human patient data. Serum proteomics coupled with least absolute shrinkage and selection operator and Boruta machine learning algorithms were used to identify protein panels predictive of postoperative sinus tachycardia. Cross-species validation was performed by comparing proteomic signatures from sheep and pediatric patients undergoing Glenn or Fontan surgery. RESULTS: Ovine Fontan animals demonstrated significant heart rate elevation beginning on postoperative day 1, peaking at postoperative day 3 (159.4±11.7 bpm versus preoperative, 105.3±10.5 bpm; P=0.0002), before trending toward baseline by postoperative day 10. This pattern was mirrored in human patients with a more modest magnitude. Surgical controls did not exhibit tachycardia. The principal component most correlated with heart rate (principal component 1: r=0.78, P=2.2×10-4) was enriched for inflammatory and neural pathways. The Boruta algorithm identified an 11-protein panel with strong predictive power (area under the receiver operating characteristic curve, 0.963). Cross-species comparison demonstrated that angiotensinogen, angiotensin-converting enzyme, and pentraxin 3 were similarly dysregulated in both species postoperatively. CONCLUSIONS: This study provides molecular evidence implicating a dysregulated neurohormonal-inflammatory axis in acute postoperative Fontan sinus tachycardia and establishes a foundation for developing targeted diagnostics and therapeutics for this complication.

Animals↗
PubMed2001

Proteome analysis of Helicobacter pylori: major proteins of type strain NCTC 11637.

Proteome analysis involves the simultaneous resolution and display of proteins produced by an organism, followed by the quantitation, characterisation and identification of these proteins. As part of an ongoing study mapping and comparing the proteins expressed by various strains of the pathogenic bacterium Helicobacter pylori, we have resolved and identified 93 of the most abundant proteins expressed by type reference strain NCTC 11637. Proteins were separated by two-dimensional gel electrophoresis and stained with Coomassie G250. Intensely-stained spots were excised and digested with trypsin, and the resulting peptides were characterised by mass spectrometry. Proteins were then identified by correlating actual peptide profiles with theoretical profiles generated from published nucleotide sequences. Ninety-three of the most intensely-stained protein spots were identified as the products of 35 genes, giving a ratio of 2.7 protein gene-products per gene. The products of the tsaA, pfr, ureA and ureB genes were amongst several proteins present in multiple isoforms. Peptide mass fingerprinting data were used to identify probable post-translational protein modifications. These results suggest that H. pylori proteins are subject to a high degree of post-translational modification. Comparative proteomics of H. pylori strains should greatly assist in investigating the pathogenic properties of this bacterium.

Bacterial Proteins↗
PubMed2004

Functional genomics studied by proteomics.

The human genome contains about 30,000 genes, each creating several transcripts per gene. Transcript structures and expression are studied by high-throughput transcriptomic techniques using microarrays. Generally, transcripts are not directly operating molecules, but are translated into functional proteins, post-translationally modified by proteolysis, glycosylation, phosphorylation, etc., sometimes with great functional impact. Proteins need to be analyzed by proteomic techniques, less suited for high-throughput. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), separating thousands of proteins has developed slowly over the past quarter of a century. This technique is now quite reproducible and suitable for differential proteomics, comparing normal and diseased cells/tissues revealing differentially regulated proteins. 2D-PAGE is combined with protein-identification methods, currently mass spectrometry (MS), which has been significantly improved over the last decade. Other proteomic techniques studying protein-protein interactions are now either established or still being developed, such as peptide or protein arrays, phage display, and the yeast two-hybrid system. The strengths and weaknesses of these techniques are discussed.

Databases, Factual↗
PubMed2006

Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox.

BACKGROUND: Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults. RESULTS: Optimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venom samples were achieved using immobilized pH gradient (IPG) gels of non-linear 3-10 pH range during the isoelectric focusing step and 10-20% gradient polyacrylamide gels in the second dimension. Software-assisted analysis of the 2-DE gels images demonstrated differences in the number and intensity of spots in juvenile, sub-adult and adult venoms. Although peptide mass fingerprinting (PMF) failed to identify even a minor fraction of spots, it allowed us to group spots that displayed similar peptide maps. The spots were subjected to a combination of tandem mass spectrometry and Mascot and MS BLAST database searches that identified several classes of proteins, including metalloproteinases, serine proteinases, lectins, phospholipases A2, L-amino oxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretory proteins. CONCLUSION: The analysis of B. atrox samples from specimens of different ages by 2-DE and mass spectrometry suggested that venom proteome alters upon ontogenetic development. We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage. The results provide insight into the molecular basis of the relation between symptomatology of snakebite accidents in humans and the venom composition. Our findings underscore the importance of the use of venoms from individual specimen at various stages of maturation for the production of antivenoms.

Journal Article↗