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

Abstract

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 β-elimination of phosphate from phospho-Ser/Thr followed by Micheal addition of ethanethiol and/or ethane-d5-thiol selectively at the vinyl moiety of dehydroalanine and dehydroamino-2-butyric acid. The method was evaluated using the model phosphoprotein α(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% α(S1)-casein and dephospho-α(S1)-casein (R2 = 0.986). Additionally, the method allowed protein identification and determination of the phosphorylation sites via MS/MS fragmentation. Copyright (C) 2000 John Wiley and Sons, Ltd.