Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography.
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.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006609:High Mobility Group Proteins / Q000302:isolation & purification*; D007455:Iodine; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D012997:Solvents; D014357:Trypsin; D014452:Ubiquitins / Q000031:analogs & derivatives / Q000302:isolation & purification*
D000596:Amino Acids; D006609:High Mobility Group Proteins; D010446:Peptide Fragments; D012997:Solvents; D014452:Ubiquitins; D007455:Iodine; D014357:Trypsin