Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography |
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| Authors: | M J Cox R Shapira K D Wilkinson |
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| Affiliation: | 1. Institute for Organic Chemistry and Centre of Biomolecular Drug Research (BMWZ), Leibniz University Hannover, Schneiderberg 38, 30167 Hannover, Germany;2. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany;3. School of Biosciences/College of Life and Enviromental Sciences, Institute of Cancer and Genomic Sciences/College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK;1. CNRS, LAAS, 7 avenue du colonel Roche, F-31400 Toulouse, France;2. Université de Toulouse, LAAS, F-31400 Toulouse, France;3. Sanofi, 195 route d’Espagne, F-31036 Toulouse, France;3. Department of Chemical Engineering, Hanyang University, Seoul, Republic of Korea |
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| Abstract: | The conditions for tryptic digestion and subsequent peptide mapping of the ATP-dependent proteolysis cofactor ubiquitin and its derivatives are described. In aqueous solution, the native ubiquitin which is composed of 76 amino acids undergoes only a single cleavage at arginine-74. Full digestion of ubiquitin was obtained in 6.5 M urea, although cleavages at lysine-33 and arginine-74 were slow. Peptide mapping was achieved by reverse-phase high-performance liquid chromatography with a C18 column using a trifluoroacetic acid/triethylamine buffer system and acetonitrile as eluants. The peptides, separated using a linear gradient, were identified by amino acid analysis. Derivatives analyzed by this method include oxidized, monoiodotyrosyl, and diiodotyrosyl ubiquitin. This technique will be useful in examining peptides of chemically modified ubiquitin with respect to extent and specificity of modification. In addition, this technique will be useful in comparing ubiquitin peptides of different organisms. |
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