Quantitative Proteomics Using ~(18)O Labeling on Target Peptides and Unlabeled Standards

摘要

A combination of mass spectrometry(MS)and ~(18)O-water labeling has been applied to absolute quantitative proteomics.We introduce a new method called quantitative analysis using unlabeled standards and isotope-labeled analytes(QAUSIA).An experimental procedure with multiple-step ~(18)O labeling is proposed to produce such ~(18)O-enriched peptides for investigation of labeling efficiency,response factor,and calibration curve.Furthermore,calibration curves employing unlabeled peptide as internal standard showed good linearity and similar slopes calculated from the mass spectra and the extracted ion chromato-grams.Subsequently,the QAUSIA strategy was implemented on the targeted peptide of ovalbumin,and the calibration slope obtained from QAUSIA using the unlabeled synthetic peptide as a standard showed very similar results to the AQUA-based approach.In addition,the concentration of the QAUSIA strategy using the targeted peptide of ovalbumin was 5.07 ± 0.14 nmol with respect to the theoretical expected value of 5.02 nmol,showing excellent accuracy.