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  • 1
    Electronic Resource
    Electronic Resource
    Proteome Analysis: Genomics via the Output Rather Than the Input Code (1997)
    Springer
    The protein journal 16 (1997), S. 537-544 
    Details
    ISSN: 1573-4943
    Keywords: Proteome ; genomics ; protein expression ; gene-product mapping ; two-dimensional gel electrophoresis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A knowledge of the ‘proteome,’ total protein output encoded by a genome, provides information on (1) if and when predicted gene products are translated, (2) the relative concentrations of gene products, and (3) the extent of posttranslational modification, none of which can be accurately predicted from the nucleic acid sequence alone. The current status of proteome analysis is reviewed with respect to some of the techniques employed, automation, relevance to genomic studies, mass spectrometry and bioinformatics, limitations, and recent improvements in resolution and sensitivity for the detection of protein expression in whole cells, tissues, or organisms. The concept of ‘proteomic contigs’ is introduced for the first time. Traditional approaches to genomic analysis call upon a number of strategies to produce contiguous DNA sequence information, while ‘proteomic contigs’ are derived from multiple molecular mass and isoelectric point windows in order to construct a picture of the total protein expression within living cells. In higher eukaryotes, the latter may require several dozen image subsets of protein spots to be stitched together using advanced image analysis. The utility of both experimental and theoretical peptide-mass fingerprinting (PMF) and associated bioinformatics is outlined. A previously unknown motif within the peptide sequence of Elongation Factor Tu from Thermus aquaticus was discovered using PMF. This motif was shown to possess potential significance in maintaining structural integrity of the entire molecule.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1026330015280
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  • 2
    Electronic Resource
    Electronic Resource
    Characterisation of basic proteins from Spiroplasma melliferum using novel immobilised pH gradients (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1393-1398 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrvlamide gel electrophoresis ; Proteome ; Spiroplasma melliferum ; Mollicutes ; Functional proteome ; Protein microsequencing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has become the method of choice for efficient separation of complex protein mixtures. Previously, analysis of the Spiroplasma melliferum proteome (protein complement of a genome) has been performed with pH 3-10 and narrow range pH 4-7 IPG gel strips. We report here on the use of novel 18 cm basic (pH 6-11) immobilised pH gradients (IPG) to increase the resolution of protein spots visible within 2-D gels. These gradients were synthesised to emulate the gradient of commercially available IPG gel strips in a 5 cm region of overlap so as to attempt construction of a more complete map of cellular protein expression. Approximately 50 additional gene products were detected from S. melliferum that were not previously well-resolved or visible using wide-range pH 3-10 IPG gel strips. Twenty-seven of these were electrotransferred to polyvinylidene difluoride (PVDF) membrane and analysed by N-terminal protein microsequencing. Protein spots with an initial peak yield of as little as 100 femtomoles (fm) were sequenced to 5-10 amino acid residues, demonstrating the importance of improved sample handling procedures and analytical technologies. Many essential metabolic enzymes were shown to have basic pI, including: glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, carbamate kinase and lactate dehydrogenase. A very basic protein (pI ≍ 11.0) was identified as uridylate kinase, an enzyme indirectly associated with pyrimidine biosynthesis and thought be absent in some members of the bacterial class Mollicutes. The advent of novel basic (pH 6-11) IPGs has allowed the visualisation of a significantly greater percentage of the ‘functional proteome’, that portion of the total protein complement of a genome actively translated within a specific time frame, on 2-D electrophoresis gels. This will aid in the characterisation of translated gene products in conjunction with genome sequencing initiatives.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180814
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  • 3
    Electronic Resource
    Electronic Resource
    Evaluation of algorithms used for cross-species proteome characterisation (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1410-1417 
    Details
    ISSN: 0173-0835
    Keywords: Bioinformatics ; Genomics ; Proteome ; Peptide-mass fingerprinting ; Amino acid composition ; Protein microsequencing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The ability to effectively search databases for the identification of protein spots from two-dimensional electrophoresis gels has become an essential step in the study of microbial proteomes. A variety of analytical techniques are currently being employed during protein characterisation. A number of algorithms used to search databases, accessible via the World Wide Web, depend upon information concerning N- and C-terminal microsequence, amino acid composition, and peptide-mass fingerprinting. The effectiveness of nine such algorithms, as well as COMBINED (software developed in this laboratory for identifying proteins across species boundaries) was examined. Fifty-four ribosomal proteins from the Mycoplasma genitalium genome, and 72 amino acyl tRNA synthetases from the Haemophilus influenzae, M. genitalium and Methanococcus jannaschii genomes were chosen for study. These proteins were selected because they represent a wide range of sequence identities across species boundaries (22.7-100% identity), as detected by standard sequence alignment tools. Such sequence variation allowed for a statistical comparison of algorithm success measured against published sequence identity. The ability of analytical techniques used in protein characterisation and associated database query programs to detect identity at the functional group level was examined for proteins with low levels of homology at the gene/protein sequence level. The significance of these theoretical data manipulations provided the means to predict the utility of data acquired experimentally for non-sequence-dependent software in proteome analysis. The data obtained also predicted that ‘sequence tagging’ of peptide fingerprints would need to be accompanied by at least 11-20 residues of amino acid sequence for it to be widely used for protein characterisation across species boundaries.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180816
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  • 4
    Electronic Resource
    Electronic Resource
    ‘Proteomic contigs’ of Mycobacterium tuberculosis and Mycobacterium bovis (BCG) using novel immobilised pH gradients (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1384-1392 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Proteome ; Mycobacterium tuberculosis ; Mycobacterium bovis (BCG) ; Immobilised pH gradient ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Tuberculosis remains a major health problem throughout the world and the failure of the existing bacille Calmette-Guérin (BCG) vaccine in recent trials has prompted a search for potential replacements. Recent advances in molecular and cell biology have cast doubts on the ability of genetic analysis alone to predict polygenic human diseases and other complex phenotypes and have therefore redirected our attention to proteome studies to complement information obtained from DNA sequencing initiatives. Novel acidic (pH 2.3-5) and basic (pH 6-11) IPG gel gradients were employed in conjunction with commercially available pH 4-7 gradients to significantly increase (fourfold) the number of protein spots previously resolved on two-dimensional (2-D) gels of Mycobacterium species. A total of 772 and 638 protein spots were observed for M. bovis BCG and M. tuberculosis H37Rv, respectively, the latter corresponding to only the pH regions 4-7 and 6-11. Of interest was the bimodal distribution observed for proteins separated from M. bovis BCG across both Mr and pH ranges. Some differences in protein expression were observed between these two organisms, contrary to what may have been expected considering the high degree of conservation in gene order and sequence similarity between homologous genes. Further work will be directed towards a more detailed analysis of these differences, so as to allow more accurate diagnosis between vaccination and active tuberculosis. The latter is of major importance to epidemiological studies and for patient management.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180813
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  • 5
    Electronic Resource
    Electronic Resource
    Proteome analysis of Spiroplasma melliferum (A56) and protein characterisation across species boundaries (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1335-1346 
    Details
    ISSN: 0173-0835
    Keywords: Proteome ; Gene-product mapping ; Spiroplasma melliferum ; Two-dimensional polyacrylamide gel electrophoresis ; Protein analysis ; Mollicutes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Spiroplasma melliferum (Class: Mollicutes) is a wall-less, helical bacterium with a genome of approximately 1460 kbp encoding 800-1000 gene-products. A two-dimensional electrophoresis gel reference map of S. melliferum was produced by Phoretix 2-D gel software analysis of eight high quality gels. The reference map showed 456 silver-stained and replicated protein spots. 156 proteins (34% of visible protein spots) from S. melliferum were further characterised by one, or a combination, of the following: amino acid analysis, peptide-mass fingerprinting via matrix assisted laser desorption ionisation-time of light (MALDI-TOF) mass spectrometry, and N-terminal protein microsequencing. Proteins with close relationship to those previously determined from other species were identified across species barriers. Thus, this study represents the first larger-scale analysis of a proteome based upon the attribution of predominantly ‘unique numerical parameters’ for protein characterisation across species boundaries, as opposed to a sequence-based approach. This approach allowed all database entries to be screened for homology, as is currently the case for studies based on nucleic acid or protein sequence information. Several proteins studied from this organism were identified as hypothetical, or having no close homolog already present in the databases. Geneproducts from major families such as glycolysis, translation, transcription, cellular processes, energy metabolism and protein synthesis were identified. Several gene-products characterised in S. melliferum were not previously found in studies of the entire Mycoplasma genitalium and Mycoplasma pneumoniae (both closely related Mollicutes) genomes. The presence of such geneproducts in S. melliferum is discussed in terms of genome size as compared with the smallest known free-living organisms. Finally, the levels of expression of S. melliferum gene-products were determined with respect to total optical itensity associated with all visible proteins expressed in exponentially grown cells.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180809
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  • 6
    Electronic Resource
    Electronic Resource
    Proteomic ‘contigs’ of Ochrobactrum anthropi, application of extensive pH gradients (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1373-1383 
    Details
    ISSN: 0173-0835
    Keywords: Proteome ; Genome ; Immobilised pH gradients ; Two-dimensional polyacrylamide gel electrophoresis ; Contiguous windows of protein expression ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The most extensive linear pH gradients yet employed in combination with two-dimensional gel electrophoresis are described, along with their application in proteome analysis. A significant proportion of the protein compliment of bacterial species is believed to be accessible using an extended linear pH gradient of 2.3 to 11.0. Protein standards with predicted isoelectric points (pI) ranging from 3.24 to 9.56 were used to confirm focusing positions with respect to the immobilised pH gradients (IPG) prior to mapping studies of Ochrobactrum anthropi. Multiple gel images were used to construct contiguous windows of protein expression (‘proteomic contigs’) within 18 cm pH gradients 2.3-5, 4-7, and 6-11 in conjunction with 15%T and 7.5%T acrylamide gels, the latter being used to resolve higher molecular weight (Mr) proteins. Each IPG had a 5 cm region of similar pH gradient overlap at pH 4-5 and pH 6-7 that was used to construct an image of protein expression characteristic of whole cell lysates. This is reminiscent of genomic sequencing initiatives whereby portions are combined to form a contiguous picture of the whole. The protein maps obtained demonstrated a means of resolving the many tens of thousands of cellular proteins likely to occur in eukarvotic systems, but also highlighted the need to further optimise protein extraction, equilibration buffers, and separation conditions of higher Mr proteins occurring at extreme pI. Theoretical 2-D protein maps were constructed for five organisms for which the total DNA sequence is now available. In all cases, higher Mr acidic and basic proteins were shown to be common.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180812
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  • 7
    Electronic Resource
    Electronic Resource
    Peptide-mass fingerprinting and the ideal covering set for protein characterisation (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1399-1409 
    Details
    ISSN: 0173-0835
    Keywords: Peptide-mass fingerprinting ; Peptide fragments ; Mass spectrometry ; Proteome ; Bioinformatics ; Endoproteinase digestion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The rules that govern the dynamics of protein characterisation by peptide-mass fingerprinting (PMF) were investigated through multiple interrogations of a nonredundant protein database. This was achieved by analysing the efficiency of identifying each entry in the entire database via perfect in silico digestion with a series of 20 pseudo-endoproteinases cutting at the carboxy terminal of each amino acid residue, and the multiple cutters: trypsin, chymotrypsin and Glu-C. The distribution of peptide fragment masses generated by endoproteinase digestion was examined with a view to designing better approaches to protein characterisation by PMF. On average, and for both common and rare cutters, the combination of approximately two fragments was sufficient to identify most database entries. However, the rare cutters left more entries unidentified in the database. Total coverage of the entire database could not be achieved with one enzymatic cutter alone, nor when all 23 cutters were used together. Peptide fragments of 〉 5000 Da had little effect on the outcome of PMF to correctly characterise database entries, while those with low mass (near to 350 Da in the case of trypsin) were found to be of most utility. The most frequently occurring fragments were also found in this lower mass region. The maximum size of uncut database entries (those not containing a specific amino acid residue) ranged from 52 908 Da to 258 314 Da, while the failure rate for a single cutter in identifying database entries varied from 10 865 (8.4%) to 23 290 (18.1 %). PMF is likely to be a mainstay of any high-throughput protein screening strategy for large-scale proteome analysis. A better understanding of the merits and limitations of this technique will allow researchers to optimise their protein characterisation procedures.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180815
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