Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus

Anal Chem. 2001 Jul 1;73(13):2836-42. doi: 10.1021/ac001404c.


A new method for proteolytic stable isotope labeling is introduced to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools or their subfractions. Two 18O atoms are incorporated universally into the carboxyl termini of all tryptic peptides during the proteolytic cleavage of all proteins in the first pool. Proteins in the second pool are cleaved analogously with the carboxyl termini of the resulting peptides containing two 16O atoms (i.e., no labeling). The two peptide mixtures are pooled for fractionation and separation, and the masses and isotope ratios of each peptide pair (differing by 4 Da) are measured by high-resolution mass spectrometry. Short sequences and/or accurate mass measurements combined with proteomics software tools allow the peptides to be related to the precursor proteins from which they are derived. Relative signal intensities of paired peptides quantify the expression levels of their precursor proteins from proteome pools to be compared, using an equation described in the paper. Observation of individual (unpaired) peptides is mainly interpreted as differential modification or sequence variation for the protein from the respective proteome pool. The method is evaluated here in a comparison of virion proteins for two serotypes (Ad5 and Ad2) of adenovirus, taking advantage of information already available about protein sequences and concentrations. In general, proteolytic 18O labeling enables a shotgun approach for proteomic studies with quantitation capability and is proposed as a useful tool for comparative proteomic studies of very complex protein mixtures.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenoviridae / classification
  • Adenoviridae / genetics*
  • Amino Acid Sequence
  • Cell Line
  • Fourier Analysis
  • Hydrolysis
  • Molecular Sequence Data
  • Oxygen Isotopes
  • Peptide Mapping
  • Proteome*
  • Species Specificity
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Viral Proteins / chemistry


  • Oxygen Isotopes
  • Proteome
  • Viral Proteins