Acid-catalyzed oxygen-18 labeling of peptides.
In enzymatic (18)O-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 (18)O label into peptides. Several synthetic peptides were dissolved in dilute acid containing 50% (v/v) H(2)(18)O and incubated at room temperature. Aliquots were removed over a period of 3 weeks and analyzed by tandem mass spectrometry (MS/MS). (18)O-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 approximately 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 (18)O-labeling studies provided that storage and analysis conditions minimize low-pH exposure times. On its own, acid-catalyzed (18)O labeling is a general tagging strategy that is an alternative to the chemical, metabolic, and enzymatic isotope-labeling schemes currently used in quantitative proteomics.
Niles
Richard
R
Department of Cell and Tissue Biology, University of CaliforniaSan Francisco, San Francisco, California 94143, USA.
Witkowska
H Ewa
HE
Allen
Simon
S
Hall
Steven C
SC
Fisher
Susan J
SJ
Hardt
Markus
M
D000143:Acids / Q000737:chemistry*; D000595:Amino Acid Sequence; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010103:Oxygen Isotopes / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D000143:Acids; D010103:Oxygen Isotopes; D010455:Peptides