Proteolytic 18O Labeling by Peptidyl-Lys Metalloendopeptidase for Comparative Proteomics

Abstract

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 ≥ 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. Keywords: comparative proteomics • ¹⁸O • Lys-N • isotope labeling • mass spectrometry • protein expression