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At least 235 records · Page 13Linked to original sources
PubMed2002

Proteome analysis of highly immunoreactive proteins of Helicobacter pylori.

BACKGROUND: Identification of the immunoreactive proteins of Helicobacter pylori is important for the development of both diagnostic tests and vaccines relating to the organism. Our aim was to determine whether there are significant differences between human IgG and IgA reactivities to individual H. pylori proteins, and whether patterns of immunoreactivity are sustained across different strains of H. pylori. METHOD: The total complement of protein from seven strains of H. pylori was resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins were transferred electrophoretically onto polyvinylene difluoride (PVDF) membranes, which were probed with sera pooled either from H. pylori-infected patients, or noninfected (control) patients. Highly immunoreactive proteins were detected using chromogenic enzyme-antibody conjugates recognising either serum IgG or IgA. These proteins were then characterised by tryptic peptide-mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Highly immunoreactive proteins were detected which were common to all seven strains, and recognised by both immunoglobulin subclasses. The proteins appear to be localised in five groups. Protein analysis established that these groups encompass multiple isoforms of chaperonin HspB (two subgroups); urease beta-subunit UreB; elongation factor EF-Tu; and flagellin FlaA. The pattern of highly immunoreactive proteins was strongly conserved across the seven strains. CONCLUSION: These results suggest that within a tightly defined region on the H. pylori proteome map there are five groups of proteins that are highly reactive to both IgG and IgA. Our analysis suggests it is unlikely that the highly immunoreactive clusters harbour any significant proteins other than isoforms of HspB, UreB, EF-Tu and FlaA, and that, with the partial exception of FlaA, these clusters are strongly conserved across all seven strains.

Bacterial Proteins↗
PubMed2001

Protein chips based on recombinant antibody fragments: a highly sensitive approach as detected by mass spectrometry.

With the human genome in a first sequence draft and several other genomes being finished this year, the existing information gap between genomics and proteomics is becoming increasingly evident. The analysis of the proteome is, however, much more complicated because the synthesis and structural requirements of functional proteins are different from the easily handled oligonucleotides, for which a first analytical breakthrough already has come in the use of DNA chips. In comparison with the DNA microarrays, the protein arrays, or protein chips, offer the distinct possibility of developing a rapid global analysis of the entire proteome. Thus, the concept of comparing proteomic maps of healthy and diseased cells may allow us to understand cell signaling and metabolic pathways and will form a novel base for pharmaceutical companies to develop future therapeutics much more rapidly. This report demonstrates the possibilities of designing protein chips based on specially constructed, small recombinant antibody fragments using nano-structure surfaces with biocompatible characteristics, resulting in sensitive detection in the 600-amol range. The assay readout allows the determination of single or multiple antigen-antibody interactions. Mass identity of the antigens, currently with a resolution of 8000, enables the detection of structural modifications of single proteins.

Antibodies↗
PubMed2002

Orderly pattern of development of the autoantibody response in (New Zealand White x BXSB)F1 lupus mice: characterization of target antigens and antigen spreading by two-dimensional gel electrophoresis and mass spectrometry.

Immunoblots of a two-dimensional PAGE-separated HL-60 cell proteomic map and mass spectrometry were combined to characterize proteins targeted by autoantibodies produced by male (New Zealand White x BXSB)F(1) (WB) mice that develop lupus and anti-phospholipid syndrome. Analysis of sera sequentially obtained from seven individual mice at different ages showed that six proteins, vimentin, heat shock protein 60, UV excision-repair protein RAD23, alpha-enolase, heterogeneous nuclear ribonucleoprotein L, and nucleophosmin, were the targets of the B cell autoimmune response, and that autoantibodies to them were synthesized sequentially in an orderly pattern that recurred in all the male WB mice analyzed: anti-vimentin first and anti-nucleophosmin last, with anti-RAD23 and anti-heat shock protein 60, then anti-alpha-enolase and anti-heterogeneous nuclear ribonucleoprotein L Abs occuring concomitantly. Anti-vimentin reactivity always appeared before anti-cardiolipin and anti-DNA Abs, suggesting that vimentin is the immunogen initiating the autoimmune process. The pattern of HL-60 proteins recognized by female WB sera differed from that of male sera, indicating that the Y chromosome-linked autoimmune acceleration gene is not an accelerator but a strong modifier of the autoimmune response. Thus, 1) combining two-dimensional PAGE and mass spectrometry constitutes a powerful tool to identify the set of Ags bound by autoantibodies present in a single serum and the whole autoantibody pattern of an autoimmune disease; 2) the diversification of the autoimmune response in male WB mice occurs in a predetermined pattern consistent with Ag spreading, and thus provides a useful model to further our understanding of the development of the autoantibody response in lupus.

Animals↗
PubMed1999

Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level.

We have investigated how the programme of protein synthesis is altered in response to a loss of calcium homoeostasis in Cos-7 cells using a differential proteome mapping approach. Exposure of the cells to the calcium ionophore A23187 or thapsigargin, or alternatively, expression of a viral glycoprotein reported to deplete intracellular calcium stores, resulted in the up-regulated expression of a characteristic set of proteins. One of these is the translationally controlled tumour protein (TCTP), a cytoplasmic protein whose expression has not previously been linked to calcium perturbation. Quantitative Northern blot assay demonstrated that steady-state mRNA abundance of TCTP was also increased under these conditions. Clamping the cytosolic calcium concentration by the introduction of bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA) into cells did not affect the increase in steady-state levels of TCTP mRNA observed in response to ionophore. Therefore depletion of endoplasmic reticulum (ER) calcium, but not elevation of the cytosolic calcium concentration, was responsible for increased transcription of the TCTP gene. However, the presence of BAPTA significantly attenuated the ionophore-mediated increase in levels of the protein. Moreover, the level of TCTP in ionophore-treated cells increased in advance of a detectable increase in the corresponding mRNA abundance. These results indicate that expression of TCTP is regulated at two distinct levels in response to the concentration of calcium in different cellular compartments. Whereas depletion of the ER store causes an increase in TCTP mRNA abundance, increased cytosolic calcium concentrations regulate gene expression at the post-transcriptional level.

Animals↗
PubMed2004

[Pediatric cardiology in the genomic era].

While congenital heart disease, cardiomyopathy, arrhythmias and acquired cardiac diseases are common causes of mortality and morbidity in infants and children, the basic underlying mechanisms of many specific pediatric cardiovascular diseases still remains undetermined. Breakthroughs in molecular genetic technology have just begun to be applied in pediatric cardiology stemming from the use of chromosomal mapping and the identification of genes involved in both the primary etiology and as significant risk factors in the development of cardiac and vascular abnormalities. This review will focus on information obtained thus far by molecular genetic analysis in the diagnosis, treatment and overall understanding of pediatric cardiovascular disease pathogenesis examining both the more prevalent congenital/inherited heart defects, arrhythmias and cardiomyopathies, as well as sporadic and acquired disorders. In addition, a survey of the pediatric cardiologist's armamentarium with regards to molecular and genetic analysis is presented highlighting the current use of molecular diagnostic methods including microarray, gene-mapping, proteomic, transgenic and stem cell technologies as well as future directions in both clinical application and research.

Cardiology↗
PubMed2006

[Stress proteins of Bifidobacterium longum NCC2705].

Stress proteins of Bifidobacterium longum strain NCC2705 were identified and characterized during stationary phase. According to the proteomic map of L. lactics IL1403 and theoretical Mr/pl of stress proteins in B. longum NCC2705 genome annotation, spots of stress proteins in gels were localized, and proteins were identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and/or ESI-MS/MS mass spectrometry. For protein identification by peptide mass fingerprinting, peptide masses were searched against database of B. longum NCC2705 by Mascot licensed in-house. 44 spots representing 8 protein entries have been identified, these proteins were hydrophilic proteins and predicted acid proteins. Spot and protein analysis revealed that post-translational modifications might be common in these proteins. Except for DnaJ, the stress proteins were encoded by genes with CAI value above 0.5, and represented a large proportion of the most abundant proteins. Moreover, the results of scavenging effects on free radicals in vitro showed that B. longum NCC2705 can inhibit fatty acid oxidation and scavenge DPPH, but they scavenge weakly active oxygen free radicals. We identified a key protein that can reverse oxidative damage to proteins and lipids: alkyl hydroperoxide reductase (ahpC, BL0615)synthesized under our experimental conditions.

Bacterial Proteins↗
PubMed2003

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli.

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.

Bacterial Proteins↗
PubMed2006

ModifiComb, a new proteomic tool for mapping substoichiometric post-translational modifications, finding novel types of modifications, and fingerprinting complex protein mixtures.

A major challenge in proteomics is to fully identify and characterize the post-translational modification (PTM) patterns present at any given time in cells, tissues, and organisms. Here we present a fast and reliable method ("ModifiComb") for mapping hundreds types of PTMs at a time, including novel and unexpected PTMs. The high mass accuracy of Fourier transform mass spectrometry provides in many cases unique elemental composition of the PTM through the difference DeltaM between the molecular masses of the modified and unmodified peptides, whereas the retention time difference DeltaRT between their elution in reversed-phase liquid chromatography provides an additional dimension for PTM identification. Abundant sequence information obtained with complementary fragmentation techniques using ion-neutral collisions and electron capture often locates the modification to a single residue. The (DeltaM, DeltaRT) maps are representative of the proteome and its overall modification state and may be used for database-independent organism identification, comparative proteomic studies, and biomarker discovery. Examples of newly found modifications include +12.000 Da (+C atom) incorporation into proline residues of peptides from proline-rich proteins found in human saliva. This modification is hypothesized to increase the known activity of the peptide.

Adult↗
PubMed2007

Sorting and pooling strategy: a novel tool to map a virus proteome for CD8 T-cell epitopes.

Human cytomegalovirus (CMV) is a major cause of morbidity in immunocompromised individuals. However, no efficient vaccine has been developed to date. Identification of T-cell target proteins and epitopes is crucial not only for developing a successful immunization strategy, but also for new approaches using adoptive transfer of antigen-specific T-cells. The CMV genome has more than 200 open reading frames potentially coding for as many proteins. Here, we describe a robust, fast, and simple SPOT synthesis strategy, which allowed us to micro-synthesize every possible CD8 T-cell epitope in the entire potential CMV proteome. So far, 9069 of these peptides have been tested in an ex vivo T-cell stimulation assay. As well as confirming a number of previously known epitopes, we identified several new ones.

Amino Acid Sequence↗
PubMed2001

Thiourea enhances mapping of the proteome from murine white adipose tissue.

Adipose tissue imposes problems in two-dimensional (2-D) analysis due to its extremely high content of fat. To improve protein separation detergents and chaotropes were varied in the IEF step. The most important factor for obtaining distinct spots in the 2-D gel was whether thiourea was included or not. Many high molecular weight spots became resolved by using thiourea, while no spots disappeared or showed inferior characteristics, thus approximately twice as many spots were possible to quantify. Hydrophobic indices were compared for a set of proteins that gave rise to sharper spots with proteins that were not improved on the use of thiourea. The comparison did not give any statistically significant difference between the two groups of proteins. One of the effects obtained by inclusion of thiourea was that the dominating protein, serum albumin, appeared as more condensed spots allowing other minor proteins to be detected. This work resulted in a protocol which greatly enhances the resolution of proteins in adipose tissue. A 2-D map of mouse white adipose tissue from epididymal fat pads was constructed in which 140 spots were identified by mass spectrometry. This work lays the ground for our further studies on white adipose tissue in metabolic diseases such as obesity and dyslipidemia.

Adipose Tissue↗
PubMed2006

Decoding 2D-PAGE complex maps: relevance to proteomics.

This review describes two mathematical approaches useful for decoding the complex signal of 2D-PAGE maps of protein mixtures. These methods are helpful for interpreting the large amount of data of each 2D-PAGE map by extracting all the analytical information hidden therein by spot overlapping. Here the basic theory and application to 2D-PAGE maps are reviewed: the means for extracting information from the experimental data and their relevance to proteomics are discussed. One method is based on the quantitative theory of statistical model of peak overlapping (SMO) using the spot experimental data (intensity and spatial coordinates). The second method is based on the study of the 2D-autocovariance function (2D-ACVF) computed on the experimental digitised map. They are two independent methods that are able to extract equal and complementary information from the 2D-PAGE map. Both methods permit to obtain fundamental information on the sample complexity and the separation performance and to single out ordered patterns present in spot positions: the availability of two independent procedures to compute the same separation parameters is a powerful tool to estimate the reliability of the obtained results. The SMO procedure is an unique tool to quantitatively estimate the degree of spot overlapping present in the map, while the 2D-ACVF method is particularly powerful in simply singling out the presence of order in the spot position from the complexity of the whole 2D map, i.e., spot trains. The procedures were validated by extensive numerical computation on computer-generated maps describing experimental 2D-PAGE gels of protein mixtures. Their applicability to real samples was tested on reference maps obtained from literature sources. The review describes the most relevant information for proteomics: sample complexity, separation performance, overlapping extent, identification of spot trains related to post-translational modifications (PTMs).

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Expression and function of proteins during development of the basal region in rice seedlings.

A differential display of proteins with a two-dimensional polyacrylamide gel electrophoresis approach was used to analyze protein expression changes during development of the basal region in rice seedlings (Oryza sativa L. cv. Nipponbare). The proteins were detected as 700 Coomassie Brilliant Blue-stained spots with pI values from around 3.5 to 9.0. A proteome reference map was established for the basal region of two-week-old seedlings. The basal region proteome map was used to analyze quantitative variations in the tissue during development from 2-, 4-, 6-, 8-, and 10-week-old seedlings. During development, 31 proteins were up-regulated, and 30 proteins were down-regulated compared with the 2-week-old basal region proteome map. The main functions of these proteins were primary metabolism and protein synthesis or maintenance. Calreticulin precursor, enolase, and voltage-dependent anion channel were identified among the up- and down-regulated proteins. The twin spots of calreticulin precursor and enolase with different pI values are possibly due to post-translational modifications such as phosphorylation. In addition, seven proteins showed developmental stage-specific expression. All of the developmentally regulated proteins of the basal region were clustered by the S-system, a differential equation that fit to time course of cluster and analyzed for cluster relationships. Proteins with unknown functions were tentatively assigned to functional groups based on cluster relationships. Basal region development proteome data will be valuable for resolving questions in functional genomics. In addition, cluster analysis of the basal region proteome during development will be useful for the assessment of functional proteins.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1999

Proteomics: quantitative and physical mapping of cellular proteins.

Genome sequencing provides a wealth of information on predicted gene products (mostly proteins), but the majority of these have no known function. Two-dimensional gel electrophoresis and mass spectrometry have, coupled with searches in protein and EST databases, transformed the protein-identification process. The proteome is the expressed protein complement of a genome and proteomics is functional genomics at the protein level. Proteomics can be divided into expression proteomics, the study of global changes in protein expression, and cell-map proteomics, the systematic study of protein-protein interactions through the isolation of protein complexes.

Biotechnology↗
PubMed2003

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis maps: relevance to proteomics.

Proteomics requires a large-scale, simultaneous separation of proteins from a mixture, assessment of the relative abundance of these molecules, and identification and characterization of each component. In 2-D PAGE separations, the best method of choice for protein analysis, separation of all the proteins present in the sample is still far to be achieved and comigrating proteins in the same spot are in general present. A statistical estimation of the degree of spot overlapping present in a 2-D PAGE separation is here described: for different conditions of spot overcrowding in the map, the degree of overlapping can be quantified in terms of purity degree of each spot or percentage of proteins that will appear in the map as a single spot. A computer simulation approach is described: it is based on the protein separation pattern present in the experimental maps. The results thus obtained are compared to a theoretical model (statistical degree of peak overlapping model) based on random spot position. The described procedures were applied to an experimental reference map of human plasma. The severity of spot overlapping in 2-D PAGE maps is estimated and the influence of different experimental conditions (strip dimension, detector system performance, pI range) is discussed. These informations are useful to quantitatively estimate the degree of error associated with identification and quantitation of each protein and to set-up experimental conditions which will increase resolution and greatly decrease the probability of spot overlapping.

Computer Simulation↗
PubMed2001

Proteome and glycosylation mapping identifies post-translational modifications associated with aggressive breast cancer.

Changes in glycosylation of glycoproteins and glycolipids is a common feature of cancer and may influence cancer cell behaviour, perhaps by enabling cell-cell interactions which favour metastasis or by allowing cancer cells to evade immuno-surveillance. Studies to identify glycosylation changes in human cancer have often used immunohistochemical techniques with lectins or antibodies and human tissue sections. Whilst some detailed biochemical studies have been performed there are few clinically relevant studies since the numbers of specimens evaluated are often very small. Using an immuno-histochemical approach, we have found that the lectin HPA from the albumen gland of Helix pomatia detects aggressive metastatic breast cancers. We have sought to identify the breast cancer associated oligosaccharides and the proteins to which they are attached via 2-DE for proteome analysis and HPLC separations for glycosylation mapping. Sixty-nine breast cancer specimens were studied. The HPA staining pattern, clinical treatment and follow-up data were known. Oligosaccharides that related to HPA staining were identified and found in elevated levels in metastatic breast cancer specimens. Human breast and colorectal cancer cells grown in vitro and in a clinically relevant animal model of metastasis also show increased levels of the oligosaccharides. We are now structurally characterising the oligosaccharides and aim to use them as diagnostic tools and targets for immuno-therapy of breast and other solid tumours.

Breast Neoplasms↗
PubMed2005

Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells.

Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress.

Animals↗
PubMed2000

From the analysis of protein complexes to proteome-wide linkage maps.

Recent advances in genomics have led to the accumulation of an unprecedented amount of data about genes. Proteins, not genes, however, sustain function. The traditional approach to protein function analysis has been the design of smart genetic assays and powerful purification protocols to address very specific questions concerning cellular mechanisms. Lately, a number of proteome-wide functional strategies have emerged, giving rise to a new field in biology, proteomics, that addresses the biology of a cell as a whole.

Molecular Biology↗