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

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