PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “differential proteomics analysis”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources

Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761.

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria's proteome was achieved using Beckman Coulter's Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.

Bacterial Proteins↗

Proteomic analysis of transcription factor interactions in myeloid stem cell development and leukaemia.

Recent results indicate that interactions of transcription factors with other nuclear proteins play an important role in stem cell development, lineage commitment and differentiation in the haematopoietic system, and the pathogenesis of myeloid leukaemias. High-throughput proteomics by mass spectrometric analysis of gel-separated proteins can identify multi-protein complexes and changes in the expression of multiple proteins simultaneously. This review describes an application of proteomic methods (2D gel electrophoresis (GE) and mass spectrometry (MS)), which can be used to identify regulated protein targets of transcription factors important in myeloid differentiation and leukaemia. This global high-throughput functional proteomics approach could lead to new insights into the network of protein-protein interactions and target proteins involved in myeloid stem cell development and leukaemia as well as provide new targets for rational pathogenesis-based therapies of leukaemia and cancer.

CCAAT-Enhancer-Binding Proteins↗

Proteomic analysis of lymph.

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.

Animals↗

Processing methods for differential analysis of LC/MS profile data.

BACKGROUND: Liquid chromatography coupled to mass spectrometry (LC/MS) has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data. RESULTS: We present a software package MZmine that enables differential LC/MS analysis of metabolomics data. This software is a toolbox containing methods for all data processing stages preceding differential analysis: spectral filtering, peak detection, alignment and normalization. Specifically, we developed and implemented a new recursive peak search algorithm and a secondary peak picking method for improving already aligned results, as well as a normalization tool that uses multiple internal standards. Visualization tools enable comparative viewing of data across multiple samples. Peak lists can be exported into other data analysis programs. The toolbox has already been utilized in a wide range of applications. We demonstrate its utility on an example of metabolic profiling of Catharanthus roseus cell cultures. CONCLUSION: The software is freely available under the GNU General Public License and it can be obtained from the project web page at: http://mzmine.sourceforge.net/.

Algorithms↗

Verification of biological markers of subacute cutaneous lupus erythematosus via TMT labelling proteomics combined with transcriptome data.

OBJECTIVE: This study aimed to investigate biological markers in subacute cutaneous lupus erythematosus (SCLE). METHODS: The tandem mass tag (TMT)-labelling proteomics method was used to explore differentially expressed proteins between SCLE lesions and normal skin tissues. The differences in transcriptomic data between SCLE tissues and normal skin tissues were analysed from the GEO database (GSE81071, GSE109248 and GSE112943). The differences in transcriptomic data from peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) and normal controls were analysed (GSE81622 and GSE154851). The 35 healthy controls, 30 SCLE patients, 35 SLE patients and 30 lupus nephritis (LN) patients were diagnosed and enrolled. The serum expression levels of IFI44 and EPSTI1 were detected. Data were presented as the mean&#xa0;&#xb1;&#xa0;standard deviation or frequency and were analysed using Student's t-test, Chi-square test and one-way ANOVA between the groups. Receiver operating characteristic (ROC) curves were used to analyse the clinical efficacy of IFI44 and EPSTI1 in distinguishing SCLE from SLE. RESULTS: In a comparative analysis of SCLE lesions and normal skin tissues, proteomics studies identified 376 proteins that exhibited significant differential expression. In GO and KEGG analyses, the enriched terms mainly included the interferon-gamma-mediated signalling pathway (p&#xa0;<&#xa0;.001), immune receptor activity (p&#xa0;<&#xa0;.001) and cell adhesion molecules (p&#xa0;<&#xa0;.001). The top 10 hub genes were screened in SCLE as follows: CD8A, CXCL10, IFI44, CD7, CCL5, TLR4, EPSTI1, ISG15, KLRD1 and SELL using Cytoscape (3.10.1) software. The 15 common proteins/genes between proteomics and three datasets results were found, including CXCL10, OAS1, DDX60L, CFB, IFI6, HERC6, IFI44L, GBP1, EPSTI1, OAS2, CXCL11, TYMP, IFI44, ISG15 and IFIT3. The 61 differentially expressed genes in GSE81622 and the top 100 differentially expressed genes in GSE154851, alongside the 15 identified genes described above through Venn diagram analysis. Four common genes, IFI44L, IFI44, EPSTI1 and OAS1, were identified. Two common genes, IFI44 and EPSTI1, were found in hub genes from the proteomics results. The serum levels of IFI44 and EPSTI1 in LN were significantly higher than those in SLE patients (p&#xa0;<&#xa0;.05). ROC curve analysis demonstrated that serum levels of IFI44 and EPSTI1 could differentiate SCLE from SLE with an area under the curve (AUC) of 0.898 and 0.847, respectively. CONCLUSIONS: The IFI44 and EPSTI1 proved to be closely involved in the progression from SCLE to SLE, and can represent new candidate diagnostic molecular markers of occurrence and progression of SCLE.

Humans↗

Differentially expressed protein markers in human submandibular and sublingual secretions.

Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

Adult↗

Proteomics of human breast ductal carcinoma in situ.

We report the first proteomic analysis of matched normal ductal/lobular units and ductal carcinoma in situ (DCIS) of the human breast. An understanding of the transition from normal epithelium to the first definable stage of cancer at the functional level of protein expression is hypothesized to contribute to improved detection, prevention, and treatment. Ten sets of two-dimensional gels were evaluated, containing either matched normal ductal/lobular units or DCIS from either whole tissue sections or up to 100,000 laser capture microdissected epithelial cells. Differential protein expression was confirmed by image analysis. Protein spots (315) were excised and subjected to mass spectrometry sequencing. Fifty-seven proteins were differentially expressed between normal ductal/lobular units and DCIS. Differences in overall protein expression levels and posttranslational processing were evident. Ten differentially expressed proteins were validated in independent DCIS specimens, and 14 of 15 proteomic trends from two-dimensional gel analyses were confirmed by standard immunohistochemical analysis using a limited independent tumor cohort. Many of the proteins identified were previously unconnected with breast cancer, including proteins regulating the intracellular trafficking of membranes, vesicles, cancer preventative agents, proteins, ions, and fatty acids. Other proteomic identifications related to cytoskeletal architecture, chaperone function, the microenvironment, apoptosis, and genomic instability. Proteomic analysis of DCIS revealed differential expression patterns distinct from previous nucleic acid-based studies and identified new facets of the earliest stage of breast cancer progression.

Breast Neoplasms↗

Plasma proteomics reveal SERPINA1 and CD59 as candidate biomarkers for COVID-19 severity stratification and prognosis prediction.

BACKGROUND: COVID-19 has been closely associated with coagulation abnormalities. However, existing biomarkers, including D-dimer and fibrin degradation products (FDP), exhibit limited accuracy in stratifying disease severity and predicting long-term clinical outcomes. OBJECTIVES: This study aimed to use proteomic analysis to identify plasma biomarkers associated with COVID-19 severity and prognosis, and validate their predictive utility for mortality and thromboembolic complications. METHODS: Plasma proteomic profiles were analyzed across three COVID-19 severity classes. Differential expression analysis and functional analysis were performed. Clustering analysis was used to identify proteins correlated with disease severity. Candidate biomarkers were validated in an independent cohort. Predictive performance of the biomarkers for mortality, sepsis and venous thromboembolism was evaluated using bootstrap-corrected ROC analyses and multivariable regression analyses. RESULTS: Proteomic analysis revealed progressive involvement of the coagulation and complement pathway with increasing disease severity. SERPINA1 and CD59 were identified as candidate biomarkers and exhibited significantly higher plasma levels in severe cases. Bootstrap-corrected ROC analyses demonstrated strong predictive performance: SERPINA1 achieved AUCs of 0.775 and 0.924 for 30-day and 12-month mortality, and CD59 achieved AUCs of 0.720 for sepsis; the combined model further improved prediction of 12-month mortality (AUC 0.946) and sepsis (AUC 0.904), outperforming D-dimer and FDP. Multivariable regression confirmed their independent prognostic value. CONCLUSION: This exploratory study identifies SERPINA1 and CD59 as candidate prognostic biomarkers in COVID-19, highlighting the role of coagulation and complement-related pathways in disease severity and warranting further prospective validation.

Humans↗

The cassava (Manihot esculenta Crantz) root proteome: protein identification and differential expression.

Using high-resolution 2-DE, we resolved proteins extracted from fibrous and tuberous root tissues of 3-month-old cassava plants. Gel image analysis revealed an average of 1467 electrophoretically resolved spots on the fibrous gels and 1595 spots on the tuberous gels in pH 3-10 range. Protein spots from both sets of gels were digested with trypsin. The digests were subjected to nanoelectrospray quadrupole TOF tandem mass analysis. Currently, we have obtained 299 protein identifications for 292 gel spots corresponding to 237 proteins. The proteins span various functional categories from energy, primary and secondary metabolism, disease and defense, destination and storage, transport, signal transduction, protein synthesis, cell structure, and transcription to cell growth and division. Gel image analysis has shown unique, as well as up- and down-regulated proteins, present in the tuberous and the fibrous tissues. Quantitative and qualitative analysis of the cassava root proteome is an important step towards further characterization of differentially expressed proteins and the elucidation of the mechanisms underlying the development and biological functions of the two types of roots.

Electrophoresis, Gel, Two-Dimensional↗

New data analysis and mining approaches identify unique proteome and transcriptome markers of susceptibility to autoimmune diabetes.

Non-obese diabetic (NOD) mice spontaneously develop autoimmunity to the insulin producing beta cells leading to insulin-dependent diabetes. In this study we developed and used new data analysis and mining approaches on combined proteome and transcriptome (molecular phenotype) data to define pathways affected by abnormalities in peripheral leukocytes of young NOD female mice. Cells were collected before mice show signs of autoimmunity (age, 2-4 weeks). We extracted both protein and RNA from NOD and C57BL/6 control mice to conduct both proteome analysis by two-dimensional gel electrophoresis and transcriptome analysis on Affymetrix expression arrays. We developed a new approach to analyze the two-dimensional gel proteome data that included two-way analysis of variance, cluster analysis, and principal component analysis. Lists of differentially expressed proteins and transcripts were subjected to pathway analysis using a commercial service. From the list of 24 proteins differentially expressed between strains we identified two highly significant and interconnected networks centered around oncogenes (Myc and Mycn) and apoptosis-related genes (Bcl2 and Casp3). The 273 genes with significant strain differences in RNA expression levels created six interconnected networks with a significant over-representation of genes related to cancer, cell cycle, and cell death. They contained many of the same genes found in the proteome networks (including Myc and Mycn). The combination of the eight, highly significant networks created one large network of 272 genes of which 82 had differential expression between strains either at the protein or the RNA level. We conclude that new proteome data analysis strategies and combined information from proteome and transcriptome can enhance the insights gained from either type of data alone. The overall systems biology of prediabetic NOD mice points toward abnormalities in regulation of the opposing processes of cell renewal and cell death even before there are any clear signatures of immune system activation.

Analysis of Variance↗

Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all-trans retinoic acid in vitro.

Embryonic stem (ES) cells are totipotent stem cells, which can differentiate into various kinds of cell types, including neurons. They are widely used as a model system for investigating mechanisms of differentiation events during early mouse development. In this study, proteomic techniques were used to approach the protein profile associated with the early-stage differentiation of ES cells into neuronal cells induced by all-trans retinoic acid (ATRA) in vitro. In comparison of the protein profile of parent ES cells with that of ES-derived neural-committed cells, which was induced by ATRA for four days, 24 differentially displayed protein spots were selected from two-dimensional electrophoresis (2-DE) gels for further protein identification by pepide mass fingerprinting (PMF). Nine proteins were known to being involved in the process of neural differentiation and/or neural survival. Of those, alpha-3/alpha-7 tubulin and vimentin were down-regulated, while cytokeratin 8, cytokeratin 18, G1/S-special cyclin D2, follistatin-related protein, NEL protein, platelet-activating factor acetylhydrolase IB alpha-subunit, and thioredoxin peroxidase 2 were upregulated during differentiation of ES cells to neural cells. Additionally, other 12 protein (five upregulated and seven downregulated) spots associated with ES cell differentiation into neuronal cells were not matched to known proteins so far, implicating that they might be novel proteins. The results above indicated that the molecular mechanisms of differentiation of ES cells to neural cells in vitro might be similar to those of other neural systems in vitro and identified that proteomic analysis is an effective strategy to comprehensively unravel the regulatory network of differentiation.

Animals↗

A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum.

Protozoan parasites of the genus Leishmania are found as promastigotes in the sandfly vector and as amastigotes in mammalian macrophages. Mechanisms controlling stage-regulated gene expression in these organisms are poorly understood. Here, we applied a comprehensive approach consisting of protein prefractionation, global proteomics and targeted DNA microarray analysis to the study of stage differentiation in Leishmania. By excluding some abundant structural proteins and reducing complexity, we detected and identified numerous novel differentially expressed protein isoforms in L. infantum. Using 2-D gels, over 2200 protein isoforms were visualized in each developmental stage. Of these, 6.1% were strongly increased or appeared unique in the promastigote stage, while the relative amounts of 12.4% were increased in amastigotes. Amastigote-specific protein isoform and mRNA expression trends correlated modestly (53%), while no correlation was found for promastigote-specific spots. Even where direction of regulation was similar, fold-changes were more modest at the RNA than protein level. Many proteins were present in multiple spots, suggesting that PTM is extensive in this organism. In several cases, different isoforms appeared to be specific to different life stages. Our results suggest that post-transcriptional controls at translational and post-translational levels could play major roles in differentiation in Leishmania parasites.

Animals↗

Selective capture of prostatic basal cells and secretory epithelial cells for proteomic and genomic analysis.

Basal cells play an undefined role in signaling the growth and differentiation of normal secretory epithelial cells in the human prostate. Because basal cells disappear during malignant transformation, we hypothesize that loss of basal cell function may have a permissive role in progression of prostate intraepithelial neoplasia into invasive carcinoma. We describe an immuno-laser capture microdissection approach to selectively capture basal cells. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we identified several protein candidates selectively expressed in microdissected basal cells. We also demonstrate that the RNA derived form this technique is an excellent source for gene-array studies. Thus, we provide evidence that proteomic and microgenomic techniques can be successfully applied to investigate the expression profiles of basal and secretory cells after immuno-capture.

Cell Transformation, Neoplastic↗

Integrated multi-omics strategies for identifying novel therapies in psoriasis.

MOTIVATION: Psoriasis is a chronic, immune-mediated disorder with an unmet need for effective treatments. To systematically prioritize therapeutic targets, we integrated proteome-wide Mendelian randomization (MR) with expression validation in blood/skin, genetic susceptibility analysis, differential gene expression (DGE) from bulk and single-cell RNA sequencing (scRNA-seq), colocalization, pathway enrichment, and protein-protein interaction analyses. RESULTS: Proteome-wide MR identified 29 candidate protein targets (Bonferroni-corrected), all replicated in independent datasets. Fifteen targets showed significant expression associations in blood or skin. Eleven proteins-UBLCP1, IL23A, ASF1A, RARRES2, ICAM1, PRSS53, ICAM5, GCA, IL2RA, DBI, and NFKB1-exhibited consistent directional effects with their genes. Genetic susceptibility analysis confirmed 20 target-specific polygenic scores for psoriasis and five for psoriatic arthritis. DGE analysis identified 13 targets in bulk and 13 in scRNA-seq-primarily in keratinocytes and immune cells-with IL2RA, COMP, and A2ML1 dysregulated across both. Colocalization analysis implicated shared causal variants for psoriasis in ASF1A, CD8A, CTF1, IL7R, MMP12, RARRES2, XCL2, DBI, IL23A, IL2RA, SGSH, and TIMD4. Enrichment analyses highlighted involvement in cytotoxicity, immune regulation, and JAK-STAT signaling. Eighteen targets interacted with approved anti-psoriasis drugs. Notably, drugs targeting IL2RA, IL7R, CTF1, ICAM1, MMP12, NFKB1, CD8A, DDX58, IL12A, SGSH, and FAP are approved or in trials for other diseases, suggesting repurposing potential. Our integrative multi-omics approach prioritized 29 high-confidence targets, including 13 novel candidates (RARRES2, ASF1A, CTF1, DBI, B3GNT2, CD8A, TIMD4, CRTAM, SGSH, XCL2, DAPK2, A2ML1, and FAP). Several high-priority targets-such as IL2RA, IL23, MMP12, RARRES2, IL7R, and ICAM1-were supported across analytical layers. These findings provide a robust foundation for psoriasis drug development. AVAILABILITY AND IMPLEMENTATION: The code used for the analyses in this manuscript has been archived in Zenodo at [DOI: 10.5281/zenodo.19692128].

Psoriasis↗

Comparative proteomic profiles of meningioma subtypes.

Meningiomas are classified into three groups (benign, atypical, and anaplastic) based on morphologic characteristics. Atypical meningiomas, which are WHO grade 2 tumors, and anaplastic meningiomas, which are WHO grade 3 tumors, exhibit an increased risk of recurrence and premature death compared with benign WHO grade 1 tumors. Although atypical and anaplastic meningiomas account for <10% of all of meningiomas, it can be difficult to distinguish them from benign meningiomas by morphologic criteria alone. We used selective tissue microdissection to examine 24 human meningiomas and did two-dimensional gel electrophoresis to determine protein expression patterns. Proteins expressed differentially by meningiomas of each WHO grade were identified and sequenced. Proteomic analysis revealed protein expression patterns unique to WHO grade 1, 2, and 3 meningiomas and identified 24 proteins that distinguish each subtype. Fifteen proteins showed significant changes in expression level between benign and atypical meningiomas, whereas nine distinguished atypical from anaplastic meningiomas. Differential protein expression was confirmed by Western blotting and immunohistochemistry. We established differential proteomic profiles that characterize and distinguish meningiomas of increasing grades. The proteins and proteomic profiles enhance understanding of the pathogenesis of meningiomas and have implications for diagnosis, prognosis, and treatment.

Electrophoresis, Gel, Two-Dimensional↗

Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma.

In order to identify new molecular markers for pancreatic intra-epithelial neoplasias (PanINs), the precursor lesions of pancreatic ductal adenocarcinoma, we established a proteomics approach analysing microdissected PanIN cells. Due to the limited amount of proteins available from microdissection, we developed a procedure including fluorescence dye saturation labelling in combination with high resolution two-dimensional gel electrophoresis. With this procedure we were able to analyse proteins extracted from 1000 microdissected cells with a high resolution of up to 2500 protein spots. Using protein lysates from the pancreatic carcinoma tissue as a reference proteome, we were able to successfully identify the proteins. Thus, we could match approximately 2200 protein spots (92%) of the microdissected sample proteome to the reference proteome for protein identification using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and nanoliquid chromatography-electrospray ionisation tandem mass spectrometry after in-gel digestion. The first proteome analysis of microdissected PanIN-2 grades revealed eight differentially expressed proteins. The differential expression of the three actin filament-associated proteins--transgelin, vimentin and MRCL3 as well as actin itself--indicates a relevant role of the actin cytoskeleton during pancreatic tumour progression. Additionally, two members of the annexin family (annexin A2 and A4) implicate a functional contribution of exocytotic and endocytotic pathways at that stage.

Adenocarcinoma↗

Differential expression of mycobacterial proteins following phagocytosis by macrophages.

Mycobacterium tuberculosis resides within the macrophages of the host, but the molecular and cellular mechanisms of survival are poorly understood. Recent evidence suggests that the attenuated vaccine strain Mycobacterium bovis BCG is both a deletion and regulatory mutant, yet retains both its immunoprotective and intra-macrophage survival potential. In an attempt to define M. bovis BCG genes expressed during interaction with macrophages, the patterns of protein synthesis were examined by both one- and two-dimensional gel electrophoresis of BCG while inside the human leukaemic macrophage cell line THP-1. This study demonstrated that BCG expresses proteins while resident inside macrophages that are not expressed during in vitro growth in culture media or under conditions of heat shock. Western blotting analysis revealed that some of the differentially expressed proteins are specifically recognized by human M. tuberculosis-infected sera. Proteome analysis by two-dimensional electrophoresis and MS identified six abundant proteins that showed increased expression inside macrophages: 16 kDa alpha-crystallin (HspX), GroEL-1 and GroEL-2, a 31.7 kDa hypothetical protein (Rv2623), InhA and elongation factor Tu (Tuf). Identification of proteins by such a strategy will help elucidate the molecular basis of the attenuation and the vaccine potential of BCG, and may provide antigens that distinguish infection with M. tuberculosis from vaccination with BCG.

Antibodies, Bacterial↗

T cell epitope definition by differential mass spectrometry: identification of a novel, immunogenic HLA-B8 ligand directly from renal cancer tissue.

In this study, we describe a differential mass spectrometric technique for the immuno-proteomic analysis of the major histocompatibility complex (MHC) peptides of a renal cell carcinoma (RCC) biopsy compared with the healthy kidney tissue of the same patient after nephrectomy. Using a stable isotope labeling approach, we could directly compare and relatively quantify 43 MHC-peptide pairs, most of which were present in similar proportions on both normal kidney and tumor. Significantly, two dominant peptides of monoisotopic masses ([M+H](+)) 973.43 u and 967.59 u, respectively, were found exclusively in the tumor sample. One of these was identified as originating from heme oxygenase-1 (HO-1), a protein involved in induction of apoptosis resistance, immuno-suppression and neoangiogenesis and reported to be up-regulated in various cancer types. Moreover, the corresponding synthetic HO-1-derived peptide was shown to be immunogenic in vitro by generation of CD8+ T cell lines with peptide-specific cytolytic activity. Thus, this peptide is an example of a differentially identified T cell epitope that could be considered as a target for immunotherapy.

Aged↗