Comparative quantification and identification of phosphoproteins using stable isotope labeling and liquid chromatography/mass spectrometry

Rapid Commun Mass Spectrom. 2000;14(18):1677-81. doi: 10.1002/1097-0231(20000930)14:18<1677::AID-RCM84>3.0.CO;2-N.


A new liquid chromatography/mass spectrometry (LC/MS) method is described for relative quantification of phosphoproteins to simultaneously compare the phosphorylation status of proteins under two different conditions. Quantification was achieved by beta-elimination of phosphate from phospho-Ser/Thr followed by Micheal addition of ethanethiol and/or ethane-d(5)-thiol selectively at the vinyl moiety of dehydroalanine and dehydroamino-2-butyric acid. The method was evaluated using the model phosphoprotein alpha(S1)-casein, for which three phosphopeptides were found after tryptic digestion. Reproducibility of the relative quantification of seven independent replicates was found to be 11% SD. The dynamic range covered two orders of magnitude, and quantification was linear for mixtures of 0 to 100% alpha(S1)-casein and dephospho-alpha(S1)-casein (R(2) = 0.986). Additionally, the method allowed protein identification and determination of the phosphorylation sites via MS/MS fragmentation.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Caseins / analysis
  • Chromatography, Liquid
  • Hydrolysis
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptides / chemistry
  • Phosphoproteins / chemistry*
  • Trypsin


  • Caseins
  • Peptides
  • Phosphoproteins
  • Trypsin