Acid-Catalyzed Oxygen-18 Labeling of Peptides

• Published in: Analytical chemistry (Washington) Vol. 81; no. 7; pp. 2804 - 2809
• Main Authors: Niles, Richard, Witkowska, H. Ewa, Allen, Simon, Hall, Steven C, Fisher, Susan J, Hardt, Markus
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.04.2009
• Subjects: Acids - chemistry; Amino Acid Sequence; Analytical biochemistry: general aspects, technics, instrumentation; Analytical chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Catalysis; Chemical reactions; Chemistry; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Isotopes; Kinetics; Mass spectrometry; Oxygen; Oxygen Isotopes - chemistry; Peptides; Peptides - chemistry; Proteomics; Spectrometric and optical methods; Staining and Labeling - methods; Substrate Specificity; Tandem Mass Spectrometry; Catalytic reaction; Isotope labelling; Peptides; Oxygen 18; Linear regression; Regression analysis; Acid catalysis; Reaction rate; Enzymatic method; Residue; Mass spectrometry MS/MS; Room temperature; Quantitative analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac802484d

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 method for introducing 18O label into peptides. Several synthetic peptides were dissolved in dilute acid containing 50% (v/v) H2 18O 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-catalyzed 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.