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  • 1
    Electronic Resource
    Electronic Resource
    Towards an automated approach for protein identification in proteome projects (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 1941-1949 
    Details
    ISSN: 0173-0835
    Keywords: Proteome ; Automation ; Two-dimensional polyacrylamide gel electrophoresis ; Mass spectrometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The development of automated, high throughput technologies for the rapid identification of proteins is essential for large-scale proteome projects. While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of matrix assisted laser desorption ionisation  -  time of flight (MALDI-TOF) mass spectra, efficient interfaces between different stages are still lacking. We report the development of a highly automated, integrated system for large scale identification of proteins separated by two-dimensional gel electrophoresis (2-DE), based on peptide mass fingerprinting. A prototype robotic system was used to image and excise 288 protein spots from an amido black stained polyvinylidene difluoride (PVDF) blot. Protein samples were enzymatically digested with a commercial automated liquid handling system. MALDI-TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with novel automated peptide mass fingerprinting database interrogation software. Using this highly automated system, we were able to identify 95 proteins on the basis of peptide mass fingerprinting, isoelectric point and molecular weight, in a period of less than ten working days. Advantages, problems, and future developments in robotic excision systems, liquid handling, and automated database interrogation software are discussed.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150191112
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  • 2
    Electronic Resource
    Electronic Resource
    Current challenges and future applications for protein maps and post-translational vector maps in proteome projects (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 830-838 
    Details
    ISSN: 0173-0835
    Keywords: Proteome ; Genome ; Two-dimensional polyacrylamide gel electrophoresis ; Post-translational modifications ; Vector maps ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170504
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  • 3
    Electronic Resource
    Electronic Resource
    Two-dimensional gel electrophoresis for proteome projects: The effects of protein hydrophobicity and copy number (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 1501-1505 
    Details
    ISSN: 0173-0835
    Keywords: Proteome ; Two-dimensional polyacrylamide gel electrophoresis ; Protein hydrophobicity ; Protein copy number ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional (2-D) gel electrophoresis is often used in proteome projects to provide a global view of the proteins expressed in any cell or tissue type. Here we have investigated the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two-dimensional gel. The average hydropathy values of all known proteins from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were calculated, thus defining the range of protein hydrophobicity and hydrophilicity in these organisms. The average hydropathy values were then calculated for a total of 427 proteins from these species, which had been identified elsewhere on 2-D gels. Strikingly, it was seen that no highly hydrophobic proteins, as defined by average hydrophobicity values, have been found to date on 2-D gel separations of whole cell lysates. A clear hydrophobicity cutoff point was seen, above which current 2-D electrophoresis methods appear not to be useful for protein separation. The effect of cellular protein copy number on a protein's presence on a 2-D gel was investigated by means of a graphical model. This model showed how variations in protein loading and copy number per cell interact to determine the quantity of a protein that will be present on a 2-D gel. Considering the current maximum in 2-D gel loading capacity, it was found that 2-D probably can not visualize or produce analytical quantities of proteins present at less than 1000 copies per cell. We conclude that further developments of 2-D electrophoresis techniques are desirable to enable the visualization and analysis of all proteins expressed by a cell or tissue.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190847
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  • 4
    Electronic Resource
    Electronic Resource
    Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 837-844 
    Details
    ISSN: 0173-0835
    Keywords: Membrane proteins ; Solubility ; Two-dimensional polyacrylamide gel electrophoresis ; Proteome ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190539
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  • 5
    Electronic Resource
    Electronic Resource
    Detailed peptide characterization using PEPTIDEMASS - a World-Wide-Web-accessible tool (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 403-408 
    Details
    ISSN: 0173-0835
    Keywords: Peptide mass fingerprinting ; Mass spectrometry ; Post-translational modifiation ; Proteome ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In peptide mass fingerprinting, there are frequently peptides whose masses cannot be explained. These are usually attributed to either a missed cleavage site during the chemical or enzymatic cutting process, the lack of reduction and alkylation of a protein, protein modifications like the oxidation of methionine, or the presence of protein post-translational modifications. However, they could equally be due to database errors, unusual splicing events, variants of a protein in a population, or artifactual protein modifications. Unfortunately the verification of each of these possibilities can be tedious and time-consuming. To better utilize annotated protein databases for the understanding of peptide mass fingerprinting data, we have written the program “PEPTIDE-MASS”. This program generates the theoretical peptide masses of any protein in the SWISS-PROT database, or of any sequence specified by the user. If the sequence is derived from the SWISS-PROT database, the program takes into account any annotations for that protein in order to generate the peptide masses. In this manner, the user can obtain the predicted masses of peptides from proteins which are known to have signal sequences, propeptides, transit peptides, simple post-translational modifications, and disulfide bonds. Users are also warned if any peptide masses are subject to change from protein iso-forms, database conflicts, or an mRNA splicing variation. The program is freely accessible to the scientific community via the ExPASy World Wide Web server, at the URL address: www.expasy.ch/www/tools.html.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180314
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  • 6
    Electronic Resource
    Electronic Resource
    The Dictyostelium discoideum proteome - the SWISS-2DPAGE database of the multicellular aggregate (slug) (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 491-497 
    Details
    ISSN: 0173-0835
    Keywords: Dictyostelium discoideum ; Two-dimensional polyacrylamide gel electrophoresis ; Proteome ; Amino acid analysis ; Isoelectric point ; Molecular weight ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2-D PAGE with immobilised pH gradients (pH 3.5-10) in the first dimension and sodium dodecyl sulfate (SDS)-PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (pI) and molecular weight (Mr) against the SWISS-PROT database with the ExPASy AAcompID tool (http://expasy.hcuge.ch/ch2d/aacompi.html). A total of 25 proteins were identified by matching against database entries for D. discoideum, and another six by crossspecies matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS-2DPAGE database.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180325
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  • 7
    Electronic Resource
    Electronic Resource
    A role for Edman degradation in proteome studies (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1068-1072 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Edman degradation ; Proteome ; Sequence tag ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two-dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottle-neck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, an N-terminal sequence tag.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180707
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