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

A new liquid chromatography/mass spectrometry (LC/MS) method is described f or relative quantification of phosphoproteins to simultaneously compare the phosphorylation status of proteins under two different conditions. Quantif ication was achieved by beta-elimination of phosphate from phospho-Ser/Thr followed by Micheal addition of ethanethiol and/or ethane-d(5)-thiol select ively 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. Reprod ucibility of the relative quantification of seven independent replicates wa s found to be 11% SD, The dynamic range covered two orders of magnitude, an d 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 M S/MS fragmentation, Copyright (C) 2000 John Wiley & Sons, Ltd.