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PubMedDate not supplied

Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms.

Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.

Bacterial Proteins↗

PubMed2004

Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry.

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.

Acetonitriles↗

PubMed2002

Applications of InterPro in protein annotation and genome analysis.

The applications of InterPro span a range of biologically important areas that includes automatic annotation of protein sequences and genome analysis. In automatic annotation of protein sequences InterPro has been utilised to provide reliable characterisation of sequences, identifying them as candidates for functional annotation. Rules based on the InterPro characterisation are stored and operated through a database called RuleBase. RuleBase is used as the main tool in the sequence database group at the EBI to apply automatic annotation to unknown sequences. The annotated sequences are stored and distributed in the TrEMBL protein sequence database. InterPro also provides a means to carry out statistical and comparative analyses of whole genomes. In the Proteome Analysis Database, InterPro analyses have been combined with other analyses based on CluSTr, the Gene Ontology (GO) and structural information on the proteins.

Amino Acid Sequence↗

PubMed2003

The diagnosis and management of pre-invasive breast disease: promise of new technologies in understanding pre-invasive breast lesions.

Array-based comparative genomic hybridization, RNA expression profiling, and proteomic analyses are new molecular technologies used to study breast cancer. Invasive breast cancers were originally evaluated because they provided ample quantities of DNA, RNA, and protein. The application of these technologies to pre-invasive breast lesions is discussed, including methods that facilitate their implementation. Data indicate that atypical ductal hyperplasia and ductal carcinoma in situ are precursor lesions molecularly similar to adjacent invasive breast cancer. It is expected that molecular technologies will identify breast tissue at risk for the development of unfavorable subtypes of invasive breast cancer and reveal strategies for targeted chemoprevention or eradication.

Breast↗

PubMed2026

Codon bias variation in Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus causes a multiplicity of human diseases acquired in community and healthcare settings alike around the globe. While most studies focus on coding changes to assess genome evolution and study genetic adaptation, interrogation of silent mutations in the form of synonymous codon usage bias is less well-studied. As such, understanding of patterns in codon bias at the gene and genome levels, and how codon bias impacts protein expression in S. aureus remains incomplete. METHODS: The codon bias of 2,565 protein encoding genes from NCTC 8325 was queried against all publicly available closed S. aureus genomes. Using public BioSample data, genomes were sorted by disease state, submitting institution, and collection site. Codon bias was assessed at the level of gene and genome using the codon adaptation index (CAI), calculated using 30S and 50S ribosomal genes. Gene set enrichment analysis was applied to determine associations between physiological functions, CAI gene scores, and interquartile ranges. CAI scores were also compared to an in vitro S. aureus proteomics database to correlate codon bias and protein expression. RESULTS: CAI scores varied within and between isolates at the gene and genome levels. Genes with ribosome-associated functions were most enriched among high CAI genes, and had low CAI interquartile ranges (IQR), suggesting selective pressure to maintain high expression of these genes across all S. aureus isolates. Genome sequences submitted by Aga Khan University Hospital, Nairobi, Kenya were most different from others. For the LAC USA 300 strain, CAI and protein expression were moderately positively correlated (cor = 0.534, p < 2.2e-16). CONCLUSIONS: Codon bias in S. aureus was shown to vary between gene, and to be a source of genetic variation between isolates; CAI and in vitro protein expression were positively correlated.

Staphylococcus aureus↗

PubMed2004

Seeing better through a MIST: evaluation of monoclonal recombinant antibody fragments on microarrays.

Automation is the key approach for genomewide and proteomewide screening of function and interaction. Especially for proteomics, antibody microarrays are a useful tool for massive parallel profiling of complex samples. To meet the requirements of antibody microarrays and to obtain a great variety of antibodies, new technologies such as phage display have partly replaced the classical hybridoma method. While the selection process for phage-displayed antibody fragments itself has been automated, the bottleneck was shifted further downstream to the identification of monoclonal binders obtained from the selections. Here, we present a new approach to reduce time, material, and waste to extend automation beyond the selection process by application of conventional microarray machinery. We were able to express recombinant antibody fragments in a single inoculation and expression step and subjected them without purification directly to an automated high-throughput screening procedure based on the multiple spotting technique (MIST). While obtaining comparable sensitivities to enzyme-linked immunosorbent assays, we minimized manual interaction steps and streamlined the technique to be accessible within the automated selection procedure.

Antibodies, Monoclonal↗

PubMed2001

High-throughput genomic and proteomic analysis using microarray technology.

BACKGROUND: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication. METHODS: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000-24,000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel. RESULTS: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82-0.99 (depending on analyte) compared with ELISAs. CONCLUSIONS: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.

Arylamine N-Acetyltransferase↗

PubMed2026

Saliva and salivary pellicle composition and proteomic profile in smokers vs. non-smokers and its effect on dental erosion.

OBJECTIVE: To analyse the salivary composition and proteomic profile of saliva and the salivary pellicle in smokers compared to non-smokers, and to examine potential differences in the erosion-protective capacity of the salivary pellicle. METHODS: Twenty-five smokers and 25 non-smokers were included. Unstimulated and stimulated saliva samples were analysed regarding flow rate, pH, buffer capacity, calcium, phosphate, fluoride, and protein content. Saliva and salivary pellicle samples were analysed by data-independent acquisition mass spectrometry (DIA-MS) for proteome profiling. In an in situ experiment, intraoral splints were loaded with bovine enamel and dentine specimens for 120 min. Pellicle-covered specimens were extraorally eroded (HCl, pH 2.3, 60 s). Calcium release was determined photometrically and compared to pellicle-free controls. RESULTS: Except for phosphate in stimulated saliva (padj.=0.003), salivary parameters were not significantly different between smokers and non-smokers. Proteome profiling detected 1759±154 proteins (cumulative 1963) in saliva, and 4262±362 proteins (cumulative 4625) in the salivary pellicle. The relative abundances of 282 (unstimulated saliva), 338 (stimulated saliva), and 4 (salivary pellicle) protein groups differed significantly between smokers and non-smokers. Functional enrichment analysis of differentially abundant human proteins revealed biological processes such as coagulation, immune response, and carcinogenic reactive oxygen species processes to be impacted by smoking. The salivary pellicle had a significant erosion-protective effect in enamel compared to the control (41.4 ± 6.3 nmol/mm2), but no differences between smokers (33.2 ± 10.6 nmol/mm2, padj.=0.001) and non-smokers (32.7 ± 8.6 nmol/mm2, padj.=0.001) were found. CONCLUSION: The proteomic profiles of both unstimulated and stimulated saliva and the salivary pellicle differ between smokers and non-smokers. CLINICAL SIGNIFICANCE: Despite the different proteomic profiles indicating a significant impact of smoking on the oral cavity, the erosion-protective capacity of the salivary pellicle of smokers and non-smokers does not differ.

Dental Pellicle↗

PubMed2003

Proteomic analysis of radiation-induced alterations in L929 cells.

In this study we examined the protein expression profiles in X-irradiated L929 cells to get insight into how mammalian cells respond to radiation-induced cell damage. L929 cells were irradiated with the dose of 6 Gy and cell lysates were collected at different time intervals (20 min, 12, 24, 36, 48 and 72 h). The extracted proteins were separated by 2-DE and quantified using computerized image analysis. Proteins exhibiting significant abundance alterations when comparing irradiated to unirradiated cells were identified by mass spectrometry. Using the proteomics approach we detected 47 proteins that exhibited a significant radiation-induced increase or decrease in the course of 72 h. From this group of spots 28 proteins were identified by mass spectrometry and of these 24 proteins exhibited minimally 2-fold differences in mean abundance values in comparison to controls. The identified proteins represent diverse sets of proteins participating either in protective and reparative cell responses or in induction of apoptosis and oncogenesis. The results document that proteomics is a useful method for unravelling the molecular mechanisms involved in cell reaction to ionizing radiation.

Animals↗

PubMed2004

Information assessment on predicting protein-protein interactions.

BACKGROUND: Identifying protein-protein interactions is fundamental for understanding the molecular machinery of the cell. Proteome-wide studies of protein-protein interactions are of significant value, but the high-throughput experimental technologies suffer from high rates of both false positive and false negative predictions. In addition to high-throughput experimental data, many diverse types of genomic data can help predict protein-protein interactions, such as mRNA expression, localization, essentiality, and functional annotation. Evaluations of the information contributions from different evidences help to establish more parsimonious models with comparable or better prediction accuracy, and to obtain biological insights of the relationships between protein-protein interactions and other genomic information. RESULTS: Our assessment is based on the genomic features used in a Bayesian network approach to predict protein-protein interactions genome-wide in yeast. In the special case, when one does not have any missing information about any of the features, our analysis shows that there is a larger information contribution from the functional-classification than from expression correlations or essentiality. We also show that in this case alternative models, such as logistic regression and random forest, may be more effective than Bayesian networks for predicting interactions. CONCLUSIONS: In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology (GO) functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed by Jansen et al. Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. We also found that the data discretizations used in the Bayesian methods decreased classification performance.

Artificial Intelligence↗

PubMed2001

Genome evolution at the genus level: comparison of three complete genomes of hyperthermophilic archaea.

We have compared three complete genomes of closely related hyperthermophilic species of Archaea belonging to the Pyrococcus genus: Pyrococcus abyssi, Pyrococcus horikoshii, and Pyrococcus furiosus. At the genomic level, the comparison reveals a differential conservation among four regions of the Pyrococcus chromosomes correlated with the location of genetic elements mediating DNA reorganization. This discloses the relative contribution of the major mechanisms that promote genomic plasticity in these Archaea, namely rearrangements linked to the replication terminus, insertion sequence-mediated recombinations, and DNA integration within tRNA genes. The combination of these mechanisms leads to a high level of genomic plasticity in these hyperthermophilic Archaea, at least comparable to the plasticity observed between closely related bacteria. At the proteomic level, the comparison of the three Pyrococcus species sheds light on specific selection pressures acting both on their coding capacities and evolutionary rates. Indeed, thanks to two independent methods, the "reciprocal best hits" approach and a new distance ratio analysis, we detect the false orthology relationships within the Pyrococcus lineage. This reveals a high amount of differential gains and losses of genes since the divergence of the three closely related species. The resulting polymorphism is probably linked to an adaptation of these free-living organisms to differential environmental constraints. As a corollary, we delineate the set of orthologous genes shared by the three species, that is, the genes that may characterize the Pyrococcus genus. In this conserved core, the amino acid substitution rate is equal between P. abyssi and P. horikoshii for most of their shared proteins, even for fast-evolving ones. In contrast, strong discrepancies exist among the substitution rates observed in P. furiosus relative to the two other species, which is in disagreement with the molecular clock hypothesis.

Archaeal Proteins↗

PubMed2005

Characterization and application of the biotin-switch assay for the identification of S-nitrosated proteins.

S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.

Animals↗

PubMed2001

Proteomic analysis of human skin fibroblasts grown on titanium: novel approach to study molecular biocompatibility.

Despite the growing use of titanium as an implant material, there is a lack of consensus on what constitutes the molecular basis of its biocompatibility. In the present study we compared the response of skin fibroblasts to two different growth supporting surfaces: commercially pure titanium (cpTi) and tissue culture polystyrene (TCPS). Proteins from extracts of whole cells and adsorbed serum were separated and identified by 2-dimensional gel electrophoresis. In all, 40 proteins (46 spots) were identified by matrix-assisted laser desorption/ionization and mass spectrometry, database searching, immunoblotting, running a standard, or a combination of these techniques. Many of the proteins collected from the two surfaces were found to derive from the serum used in the culture medium. The surface properties of titanium appeared to promote the formation of a more concentrated carpet of serum proteins. Several proteins from bovine or human serum, such as albumin, alpha2-HS-glycoprotein, alpha-fetoprotein, plasminogen, thrombospondin 1, and serotransferrin, along with a few unidentified serum components, were found to adsorb onto cpTi in comparatively high concentrations. The adsorption of serum proteins did not appear to be selective on either substrate. We found that among the major cellular proteins, fibronectin and a cytoskeletal protein (non-muscle myosin heavy chain type A) were expressed at lower levels by fibroblasts grown on cpTi compared to TCPS. By analyzing the changes in the entire proteome of cells in response to different growth substrates, we may gain a better understanding of the molecular basis of biocompatibility.

Animals↗

PubMed2006

Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders.

Biomarkers are needed to assist in the diagnosis and medical management of various neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body (DLB). We have employed a multiplex quantitative proteomics method, iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification), in conjunction with multidimensional chromatography, followed by tandem mass spectrometry (MS/MS), to simultaneously measure relative changes in the proteome of cerebrospinal fluid (CSF) obtained from patients with AD, PD, and DLB compared to healthy controls. The diagnosis of AD and DLB was confirmed by autopsy, whereas the diagnosis of PD was based on clinical criteria. The proteomic findings showed quantitative changes in AD, PD, and DLB as compared to controls; among more than 1,500 identified CSF proteins, 136, 72, and 101 of the proteins displayed quantitative changes unique to AD, PD, and DLB, respectively. Eight unique proteins were confirmed by Western blot analysis, and the sensitivity at 95% specificity was calculated for each marker alone and in combination. Several panels of unique makers were capable of distinguishing AD, PD and DLB patients from each other as well as from controls with high sensitivity at 95% specificity. Although these preliminary findings must be validated in a larger and different population of patients, they suggest that a roster of proteins may be generated and developed into specific biomarkers that could eventually assist in clinical diagnosis and monitoring disease progression of AD, PD and DLB.

Alzheimer Disease↗

PubMed2006

Modification of host lipid raft proteome upon hepatitis C virus replication.

Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts. We used two proteomics approaches to characterize the protein content of lipid rafts isolated from Huh7 cells and its modification upon HCV replication. Using two-dimensional electrophoresis and mass spectrometry, we identified approximately 100 protein spots in the isolated lipid rafts; among them, 39 were reproducibly modified in HCV replicon cell lines as compared with control cell lines. We also used stable isotope labeling by amino acids in cell culture (SILAC) combined with one-dimensional electrophoresis separation and mass spectrometry. Using this approach, we identified 1036 individual proteins based on peptides selected with at least 95% confidence; among them, 413 proteins were identified with at least two peptides. Quantification analysis identified 150 proteins modified by at least 2.5-fold (110 up-regulated and 40 down-regulated) in HCV-replicating cells compared with controls. Protein identifications and quantifications obtained by both proteomics approaches were largely concordant. Modulated proteins included a majority of proteins involved in vesicular and protein trafficking and in cell signaling. Remarkably for a large number of proteins, their up-regulation in lipid rafts of HCV replicon cells was due to their relocalization. By using small interfering RNAs directed to the modulated small GTPases Cdc42 and RhoA, we observed an increase in HCV replication, whereas reduction of syntaxin 7 expression resulted in decreased replication of HCV. Our findings indicate that protein subcellular relocalization occurs in HCV-containing cells that can directly affect HCV replication.

Cell Extracts↗

PubMed2003

Proteome analysis of Escherichia coli W3110 expressing an heterologous sigma factor.

The recombinant plasmid pASK18 carries a Streptomyces DNA fragment which includes an open reading frame, designated psfS (putative sigma factor, Streptomyces), as its putative product showed a high degree of similarity with RNA polymerase sigma factors. Previous results showed that PsfS causes transcription initiation within the bgl operon promoter-silencer region in Escherichia coli K12. In this study a proteomic approach has been applied in order to perform a comparative analysis of E. coli K12 W3110 wild-type, W3110 (pASK18) and a W3110 Bgl(+) spontaneous mutant. Either by qualitative or quantitative analysis, no significant difference was observed between the proteomes of W3110 and its Bgl+ derivative, while W3110 (pASK18) showed an altered profile by both analyses. Fourteen out of the 37 protein spots showing a different expression level in E. coli W3110 harboring pASK18 were identified by peptide mass fingerprinting. Among the proteins identified, thiol peroxidase (Tpx) was the only one up-regulated. The possible involvement of bgl and tpx in the survival of the pathogen E. coli during infection is discussed.

Amino Acid Sequence↗

PubMedDate not supplied

Gene microarray study corroborates proteomic findings in rodent islet cells.

As reported in an issue of Journal of Proteome Research, mass spectrometry has been used to identify numerous proteins in pancreatic islets. Our group studies beta cell gene expression, and we were interested in whether proteins described in this study could be found at the level of the transcriptome. Microarray analysis is a powerful technique for quantitative measurements of the expression of thousand genes in parallel. However, in crude tumor biopsies, only a subset of transcripts correlate with protein levels, and it is still unknown how frequently mRNA expression correlates with amount of protein in well-differentiated cells. To address this issue, we presently compared data from mouse primary islet proteins obtained by proteomic analysis with RNA data from FACS purified primary rat beta cells obtained by microarray analysis (Rasschaert J, Liu D, Cardozo AK, Kutlu B, Eizirik DL, manuscript in preparation).

Animals↗

PubMedDate not supplied

Proteomics: techniques and applications in cancer research.

Proteomics can be defined as functional analysis of the full set of proteins by high-throughput technologies in a given system. The workflow of proteomics is a multi-step process comprising sample preparation, separation, quantification and identification of proteins. Due to the high complexity of different protein species and the wide dynamic range of protein amount within a cell system it is necessary to apply appropriate analysis methods. One approach is to separate proteins first by two-dimensional gel electrophoresis (2-DE) according to charge and molecular weight. Proteins are then fragmented and analyzed using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Identification of proteins can be achieved by comparing the mass/charge-ratios of these peptides to respective databases. Proteome analysis with respect to the identification of disease-associated patterns of molecules in different tissues is in the early stages, because standardisation of these techniques often remains to be established. However, proteome analyses is a promising tool to obtain holistic insights into the physiological status of a cell or cellular system. Compared to RNA-based studies some advantages are obvious: (1) post-translational modifications, e. g. phosphorylation, contributing to the activity status can be detected at the protein level only, (2) RNA-levels do not necessarily coincide with protein levels for a particular gene, (3) feedback-mechanisms within regulatory pathways can control protein activity without measurable changes in mRNA content.

Algorithms↗