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  • 1
    Electronic Resource
    Electronic Resource
    S-Pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: A rapid mass spectrometric method for identifying cysteine-containing peptides (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 907-917 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional gel polyacrylamide gel electrophoresis ; Mass spectrometry ; Peptide mass fingerprinting ; Capillary liquid chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In-gel proteolytic digestion of acrylamide-gel separated proteins is a method widely used for generating peptide fragments for the purpose of identifying proteins by Edman degratation, tandem mass spectrometry, and peptide-mass fingerprinting. However, it is well recognised for disulfide-bonded proteins electrophoresed under reducing conditions that if no precautions are taken to minimise disulfide bond formation during protein digestion or peptide isolation, complex peptide maps can result. Here, we describe an improved method for in-gel protein digestion. It consists of first reducing and S-pyridylethylating Coomassie Brilliant Blue R-250-stained proteins immobilised in the whole gel slab with dithiothreitol and 4-vinylpyridine, excising the individual stained and alkylated proteins, and then digesting them in situ in the gel matrix with trypsin or Achromobacter lyticus protease I. Peptide fragments generated in this manner are extracted from the gel piece and purified to homogeneity by a rapid (≤12 min) reversed-phase high performance liquid chromatography (HPLC) procedure, based upon conventional silica supports. Recoveries of peptides are increased by S-pyridylethylation of acrylamide-immbilised proteins prior to in-gel digestion. Further, the levels of gel-related contaminants, which otherwise result in suppression of sample signals during electrosprayionisation mass spectrometry, are greatly reduced by the reduction / alkylation step. Additionally, we demonstrate that S-β-(4-pyridylethyl)-cysteine containing peptides can be readily identified during reversed-phase HPLC by absorance at 254 nm, and during electrospray ionisation tandem mass spectrometry by the appearance of a characteristic-pyridylethyl fragment ion of 106 Da. The position of cysteine residues in a sequence can be determined as phenylthiohydantoin S-β-(4-pyridylethyl)-cysteine during Edman degradation, and by tandem mass spectrometry.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170512
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  • 2
    Electronic Resource
    Electronic Resource
    Nonreducing two-dimensional polyacrylamide gel electrophoretic analysis of human colonic proteins (1995)
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 1120-1130 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Mass spectrometry ; Peptide mass fingerprinting ; Capillary liquid chromatography ; Human colonic proteins ; Immunoblot analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Immunochemical detection of proteins with antigenic determinants that are dependent on the native spatial conformation of the protein can often pose problems with conventional two-dimensional polyacrylamide gel electrophoresis (2-DE). For example, many antigenic determinants are readily destroyed by reducing agents and/or urea, reagents which are critical components of many of the conventional isoelectric focusing and immobilized-pH-gradient (IPG) protocols used in the first electrophoretic dimension. Here we describe the use of commercially available precast 2-DE gels for performing nonreducing/non-urea 2-DE of proteins extracted from the human colon cancer cell line LIM 1215 with 0.3% Triton X-100 that permit the identification of antigens with conformational determinants by immunoblot analysis. Previous, related studies demonstrated the usefulness of peptide-mass fingerprinting for identifying 2-DE resolved proteins. Here we show how partial protein sequence data obtained by rapid peptide mapping, using capillary column liquid chromatography directly coupled with electrospray ionization tandem mass spectrometric methodologies, enhances the usefulness of this approach for identifying incompletely resolved proteins. The nonreducing 2-DE gel images reported in this study, along with our master 2-DE gel protein database for both normal human colonic crypts and several colon-cancer-derived cell lines, and information regarding microtechniques employed in this laboratory for obtaining structural data on 2-DE resolved proteins can be accessed over the Internet using World Wide Web (URL address: http://www.ludwig.edu.au).
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501601189
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  • 3
    Electronic Resource
    Electronic Resource
    A two-dimensional gel database of human colon carcinoma proteins (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 605-613 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Tandem mass spectrometry ; Peptide mass fingerprinting ; Post-source-decay fragmentation ; Human colonic proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The master two-dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883-891; Ji et al., Electrophoresis 1994, 15, 391-405). Updated two-dimensional electrophoretic (2-DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2-DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their Mr/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2-DE resolved proteins. This procedure relies on a combination of capillary column (0.10-0.32 mm internal diameter) reversed-phase HPLC peptide mapping of in-gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision-induced dissociation or post-source-decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro-techniques employed in this laboratory for identifying 2-DE resolved proteins can be accessed via the internet URL: http://www.ludwig.edu.au.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180344
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