Proteolytic 18O labeling by peptidyl-Lys metalloendopeptidase for comparative proteomics

The potential capabilities of a new proteolytic 18O labeling method employing peptidyl-Lys metalloendopeptidase (Lys-N) have been demonstrated for use in comparative proteomics. Conditions (pH>or=9.5) have been found such that Lys-N incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide. This 18O labeling method has a major advantage over current protelytic 18O labeling methods that generate a mixture of isotopic isoforms resulting from the incorporation of one or two 18O atoms into each peptide species by the proteases (trypsin, Lys-C, or Glu-C) used. We demonstrate that the single 18O atom incorporation property of Lys-N overcomes the major problem of the current proteolytic 18O labeling methods and provides accurate quantification results for isotopically labeled peptides.