Acid-Catalyzed Oxygen-18 Labeling of Peptides for Proteomics Applications

摘要

In enzymatic ¹⁸O-labeling strategies for quantitative proteomics, the exchange of carboxyl oxygens at low pH is a common, undesired side reaction. We asked if acid-catalyzed back exchange could interfere with quantitation and whether the reaction itself could be used as an alternative method for introducing ¹⁸O label into peptides. Several synthetic amino acid sequences were dissolved in dilute acid containing 50% (v/v) H2¹⁸O and incubated at room temperature. Aliquots were removed over a period of 3 weeks and analyzed by tandem mass spectrometry (MS/MS). ¹⁸O-incorporation ratios were determined by linear regression analysis that allowed for multiple stable isotope incorporations. At low pH, peptides exchanged their carboxyl oxygen atoms with the aqueous solvent. The isotope patterns gradually shifted to higher masses until they reached the expected binomial distribution at equilibrium after ~11 days. Reaction rates were residue and sequence specific. Due to its slow nature, the acid-based back exchange is expected to minimally interfere with enzymatic ¹⁸O-labeling studies provided that storage and analysis conditions minimize low pH exposure times. On its own, acid-catalyzed ¹⁸O labeling is a general tagging strategy that is an alternative to the chemical, metabolic and enzymatic isotope-labeling schemes currently used in quantitative proteomics.