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Results for “proteome mapping”

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At least 487 records · Page 27Linked to original sources
PubMed2004

Proteomic analysis of multiple sclerosis cerebrospinal fluid.

Two-dimensional gel electrophoresis and peptide mass fingerprinting were used to identify proteins in cerebrospinal fluid (CSF) pooled from three patients with multiple sclerosis (MS) and in CSF pooled from three patients with non-MS inflammatory central nervous system (CNS) disorders. Resolution of CSF proteins on three pH gradients (3-10, 4-7 and 6-11) enabled identification of a total of 430 spots in the MS CSF proteome that represented 61 distinct proteins. The gels containing MS CSF revealed 103 protein spots that were not seen on control gels. All but four of these 103 spots were proteins known to be present in normal human CSF. The four exceptions were: CRTAC-IB (cartilage acidic protein), tetranectin (a plasminogen-binding protein), SPARC-like protein (a calcium binding cell signalling glycoprotein), and autotaxin t (a phosphodiesterase). It remains unknown whether these four proteins are related to the cause and pathogenesis of MS.

Adult↗
PubMed2006

Characterization of the proteins of bacterial strain isolated from contaminated site involved in heavy metal resistance--a proteomic approach.

The present study describes response of a bacterial strain isolated from a polluted river to heavy metal toxicity. The bacterium was identified to be Klebsiella pneumoniae by biochemical tests using API 20E strips and 16S ribotyping. The isolate was studied for its tolerance to two heavy metals, i.e., cobalt (Co(2+)) and lead (Pb(2+)) by growing it in citrate mineral medium (CMM). Proteomic approach involving two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS) was used to identify the differentially expressed proteins under heavy metal stress. Two of the differentially expressed proteins were identified to be l-isoaspartate protein carboxymethyltransferase type II and DNA gyrase A. To our knowledge, this is for the first time that K. pneumoniae has been reported to be present in metal contaminated site and l-isoaspartate protein carboxymethyltransferase type II protein to be over expressed under heavy metal stress. The role of these proteins in metal tolerance is discussed.

Bacterial Proteins↗
PubMed2004

Immunoproteomics.

Two-dimensional electrophoresis results in an adequate resolution of the proteome of microorganisms to allow the detection and identification of specific antigens after blotting on membranes and overlaying the protein pattern with patient's sera. The complement of all identified antigens presents the immunoproteome of a microorganism. All the antigens specific for a microorganism or even for a disease are identified by mass spectrometry. For identification, peptide mass fingerprinting is used, and post-translational modifications are detected by mass spectrometry MS/MS techniques. High-resolution two-dimensional electrophoresis and unambiguous identification are prerequisites for reliable results. After statistical analysis, the resulting antigens are candidates for diagnosis or vaccination and targets for therapy.

Antibodies, Bacterial↗
PubMed2006

Changes in enzymes associated with energy metabolism during the early post mortem period in longissimus thoracis bovine muscle analyzed by proteomics.

Changes in metabolic protein levels in biopsies during the early post mortem period in the bovine longissimus thoracis muscle were investigated by 2-DE based proteome analyses. Nine NRF (Norwegian Red) dual purpose bulls were included in the study. Twenty-four proteins underwent changes between the two sampling times and were classified into two major groups: metabolic proteins and heat shock proteins. Of the metabolic proteins, 5 enzymes involved in the glycolytic pathway and the tricarboxylic acid (TCA) cycle, increased in intensities during the post mortem period. In addition, the NADP-dependent enzyme 3-hydroxyisobutyrate dehydrogenase, associated with the TCA cycle in muscle, was increased. This documents that an increased aerobic energy metabolism occurs immediately after slaughter, with the aim to replenish the ATP levels in the muscle.

Animals↗
PubMed2006

Comparative proteome analysis of laboratory grown Brucella abortus 2308 and Brucella melitensis 16M.

Brucella species are pathogenic agents that cause brucellosis, a debilitating zoonotic disease that affects a large variety of domesticated animals and humans. Brucella melitensis and Brucella abortus are considered major health threats because of their highly infectious nature and worldwide occurrence. The availability of the annotated genomes for these two species has allowed a comparative proteomics study of laboratory grown B. melitensis 16M and B. abortus 2308 by two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting. Computer-assisted analysis of the different 2-D gel images of strains 16M and 2308 revealed significant quantitative and qualitative differences in their protein expression patterns. Proteins involved in membrane transport, particularly the high affinity amino acids binding proteins, and those involved in Sec-dependent secretion systems related to type IV and type V secretion systems, were differentially expressed. Differential expression of these proteins may be responsible for conferring specific host preference in the two strains 2308 and 16M.

Animals↗
PubMed2005

Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations.

Researchers have several options when designing proteomics experiments. Primary among these are choices of experimental method, instrumentation and spectral interpretation software. To evaluate these choices on a proteome scale, we compared triplicate measurements of the yeast proteome by liquid chromatography tandem mass spectrometry (LC-MS/MS) using linear ion trap (LTQ) and hybrid quadrupole time-of-flight (QqTOF; QSTAR) mass spectrometers. Acquired MS/MS spectra were interpreted with Mascot and SEQUEST algorithms with and without the requirement that all returned peptides be tryptic. Using a composite target decoy database strategy, we selected scoring criteria yielding 1% estimated false positive identifications at maximum sensitivity for all data sets, allowing reasonable comparisons between them. These comparisons indicate that Mascot and SEQUEST yield similar results for LTQ-acquired spectra but less so for QSTAR spectra. Furthermore, low reproducibility between replicate data acquisitions made on one or both instrument platforms can be exploited to increase sensitivity and confidence in large-scale protein identifications.

Algorithms↗
PubMed2004

Technical aspects of gel-based proteomics designed for elucidating an aryl hydrocarbon receptor complex.

The identification of proteins by mass spectrometry has revolutionalized the basic method of identifying proteins constituting an intracellular unit or network for certain biological functions. The gel-based strategy following immunoprecipitation was applied to elucidating proteins associated with the aryl hydrocarbon receptor (AhR). Two hundred femtomoles of AhR was recovered from approximately 2 x 10(7) HepG2 cells by immunoprecipitation and was sufficient for identification by peptide mass fingerprinting. Possible candidates for the AhR-associated proteins were also identified. Improvements of the current strategy to increase the overall sensitivity tenfold are required to clarify the AhR complex in full detail. For example, a combination of trypsin and Achromobacter protease I for in-gel digestion allows the number of missed cleavage sites to be set at zero for database searching, thereby reducing random matches and facilitating identification. There is also room for improvement in each step of sample preparation prior to mass spectrometry.

Cell Line↗
PubMed2003

A proteomics approach to understanding protein ubiquitination.

There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.

Amino Acid Sequence↗
PubMed2004

Proteomic analysis of lung biopsies: Differential protein expression profile between peritumoral and tumoral tissue.

The cellular proteome shows a dynamic profile and is subjected to changes in response to various stimuli and disease progression. Lung cancer remains the leading cause of cancer death in industrialized countries. In an attempt to find new disease markers, patients suffering from lung carcinoma have been selected to achieve differential protein expression patterns between normal and neoplasic tissue. After two-dimensional electrophoresis, the spots of interest were digested and identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting. This information will provide a more comprehensive understanding of the disease progression and might constitute a method to complement histopathological diagnosis.

Databases, Protein↗
PubMed2001

Back to the future: the human protein index (HPI) and the agenda for post-proteomic biology.

The effort to produce an index of all human proteins (the human protein index, or HPI) began twenty years ago, before the initiation of the human genome program. Because DNA sequencing technology is inherently simpler and more scalable than protein analytical technology, and because the finiteness of genomes invited a spirit of rapid conquest, the notion of genome sequencing has displaced that of protein databases in the minds of most molecular biologists for the last decade. However, now that the human genome sequence is nearing completion, a major realignment is under way that brings proteins back to the center of biological thinking. Using an influx of new and improved protein technologies--from mass spectrometry to re-engineered two-dimensional (2-D) gel systems, the original objectives of the HPI have been expanded and the time frame for its execution radically shortened. Several additional large scale technology efforts flowing from the HPI are also described.

Biotechnology↗
PubMed2001

Chromatographic separations as a prelude to two-dimensional electrophoresis in proteomics analysis.

Current methods of proteome analysis rely almost solely on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by the excision of individual spots and protein identification using mass spectrometry (MS) and database searching. 2-D PAGE is denaturing in both dimensions and, thus, cannot indicate functional associations between individual proteins. Moreover, less abundant proteins are difficult to identify. To simplify the proteome, and explore functional associations, nondenaturing anion exchange column chromatography was used to separate a soluble protein extract from Escherichia coli. Successive fractions were then analysed using 2-D PAGE and selected spots from both the gels for the start material and the fractionated material were quantified and identified by peptide mass fingerprinting using a MALDI-TOF mass spectrometer. Enrichments of up to 13-fold were attained for individual protein spots and peptide mass fingerprints were of significantly higher quality after chromatographic separation. The marked anomalies between predicted p/and column elution position contrasted with the almost perfect correlation with migration distance on isoelectric focusing (IEF) and were explored further for basic proteins.

Chromatography, Ion Exchange↗
PubMed2005

Proteomic and transcriptomic characterization of interferon-alpha-induced human primary T helper cells.

Interferon-alpha (IFN-alpha) is a multifunctional cytokine that modulates immune response. In spite of the numerous comprehensive studies on the effects of IFN-alpha on various cell types, novel characteristics of this versatile agent emerge continuously. In the present study a differential proteomic approach was used to identify new IFN-alpha-regulated proteins in human primary CD4(+) T cells. Two IFN-alpha-inducible proteins, soluble N-ethylmaleimide-sensitive factor attachment protein alpha (alpha-SNAP) and cleavage stimulation factor-64 (CstF-64) previously not described in this context, were identified. Additionally, several proteins already known as IFN-stimulated genes were observed. The results of proteomics experiments were further studied at the mRNA level using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Both peripheral blood and cord blood CD4(+) T cells were used in order to see if there are differences in IFN-alpha response between these populations. Differences were observed between the IFN-alpha-induced expression kinetics in peripheral blood and cord blood transcripts. The induction was more rapid in peripheral blood than in cord blood cells. CstF-64 expression was upregulated by IFN-alpha at the protein, but not at the mRNA level.

Autoradiography↗
PubMed2005

Proteomic analysis of Deinococcus radiodurans recovering from gamma-irradiation.

In order to reveal the mechanisms of the extreme radioresistance and DNA repair in Deinococcus radiodurans, we examined proteome changes in a wild-type strain following gamma-irradiation using two-dimensional polyacrylamide gel electrophoresis and Silver-staining. The expression levels of 26 protein spots showed significant changes under radiation stress. Of these spots, 21 were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry after tryptic in-gel digestion. These proteins exhibited various cellular functions, including (i) translation; (ii) transcription; (iii) signal transduction; (iv) post-translational modification, protein turnover, chaperones; (v) carbohydrate transport and metabolism; (vi) energy production and conversion; (vii) nucleotide transport and metabolism; (viii) inorganic ion transport and metabolism; (ix) DNA replication, recombination and repair; and (x) yet unknown. Most of the proteins have not previously been reported to be relevant to radioresistance.

Bacterial Proteins↗
PubMed2006

Proteome analysis of sorbitol fermentation specific protein in Vibrio cholerae by 2-DE and MS.

Vibrio cholerae can be differentiated into epidemic and non-epidemic strains by sorbitol fermentation speed, but little research has been done on its mechanisms. In this study, we investigated differential protein expression of the two strains in response to sorbitol metabolism. V. cholerae strains were cultured in media with and without sorbitol, respectively. Proteins were separated by 2-DE, and those that showed different expression in the two media were identified by MALDI-TOF MS. Fifteen proteins in epidemic strains and 11 proteins in non-epidemic strains showed a different expression in sorbitol medium. Among them, 4 proteins were common to epidemic and non-epidemic strains. Gene sequence analysis showed that some mutations occurred in these proteins between the two strains. Potential functions of these proteins included sugar uptake, amino acid uptake, electron transport, sulfate and thiosulfate transport.

Amino Acid Sequence↗
PubMed2006

Usefulness of an integrated microfluidic device (HPLC-Chip-MS) to enhance confidence in protein identification by proteomics.

Nanoflow liquid chromatography/mass spectrometry (nanoLC/MS) has become a current tool in proteomics applications increasingly used in the search for new biomarkers. A new integrated microfluidic device (HPLC-Chip), coupled to ion trap mass spectrometry (ITMS), appears as an innovative and robust tool for improving the identifications commonly performed by nanoLC/MS/MS. We tested this device for the identification of proteins obtained from two-dimensional gel electrophoresis or chromatography. The chip allows the measurement of reproducible retention times that, in association with m/z ratios, was found useful for identifying peptide sequences without ambiguity. A sensitivity increase of a factor of at least 5-fold is obtained compared to the results obtained previously in our laboratory by conventional nanoLC/MS/MS on the same ion trap. We conclude that this recently available microfluidic device can be a valuable tool during biomarker discovery programs, particularly identifying low-abundance proteins.

Chromatography, High Pressure Liquid↗
PubMed2006

Guanidination chemistry for qualitative and quantitative proteomics.

The application of guanidination chemistry, the conversion of lysine into homoarginine residues, is used to illustrate several important general considerations relating to the use of differential isotope labelling for relative quantification in proteomics. The derivatisation procedure has been optimised for automation using a liquid handling station designed for proteomics. Automated application of the procedure to the analysis of in-gel tryptic digests of multiple spots from the two-dimensional gel electrophoretic (2DE) analysis of proteins from the FDCP-mix cell line shows near-universal improvement in protein identification as a result of derivatisation. This chemistry has been extended for relative quantification, applicable to matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and also tandem mass spectrometry (MS/MS). It provides a robust method for the quantitative comparison of two samples that have been separated by 2DE. A peptide pair may display poor detection during MS analysis, causing their reliable relative quantification to be difficult. In such circumstances, the additional selectivity of detection provided by MS/MS can substantiate identification and allow relative quantification of these species via product ion signal ratios.

Animals↗
PubMed2006

Identification of the first archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix.

The archaeon Aeropyrum pernix grows optimally at 90 degrees C and derives energy primarily from aerobic degradation of complex proteinaceous substrates. The ability of these nutrients to sustain growth is generally associated with the presence of oligopeptide transport systems, such as the well-known protein-dependent ATP-binding cassette (ABC) transporters. This study is concerned with the isolation and characterisation of the first archaeal oligopeptide-binding protein (OppA(Ap)) from the extracellular medium of A. pernix. The protein shows a pI of 3.9 and a molecular mass of about 90 kDa under native conditions. By using a proteomic approach, the OppA(Ap)-encoding gene was identified (APE1583) and about 55% of the protein amino-acid sequence was validated. The extracellular purified protein was able to efficiently bind oligopeptide substrates such as Xenopsin. The amount of a liganded peptide to OppA(Ap) was about 70% at 90 degrees C using a 1/100 (w/w) OppA(Ap)/substrate ratio. Sequence comparisons showed a weak but significant similarity of OppA(Ap) with bacterial oligopeptide binding proteins. Furthermore, APE1583 neighbouring genes encode for the cognate components of an ABC transport system, suggesting that these ORFs are organised in an operon-like structure, with OppA(Ap )as the extracellular component for the uptake of oligopeptides.

ATP-Binding Cassette Transporters↗
PubMed2005

Quality classification of tandem mass spectrometry data.

UNLABELLED: Peptide identification by tandem mass spectrometry is an important tool in proteomic research. Powerful identification programs exist, such as SEQUEST, ProICAT and Mascot, which can relate experimental spectra to the theoretical ones derived from protein databases, thus removing much of the manual input needed in the identification process. However, the time-consuming validation of the peptide identifications is still the bottleneck of many proteomic studies. One way to further streamline this process is to remove those spectra that are unlikely to provide a confident or valid peptide identification, and in this way to reduce the labour from the validation phase. RESULTS: We propose a prefiltering scheme for evaluating the quality of spectra before the database search. The spectra are classified into two classes: spectra which contain valuable information for peptide identification and spectra that are not derived from peptides or contain insufficient information for interpretation. The different spectral features developed for the classification are tested on a real-life material originating from human lymphoblast samples and on a standard mixture of 9 proteins, both labelled with the ICAT-reagent. The results show that the prefiltering scheme efficiently separates the two spectra classes.

Algorithms↗