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 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.
Weckwerth
W
W
Max-Planck-Institute of Molecular Plant Physiology, 14424 Potsdam, Germany. weckwerth@mpimp-golm.mpg.de
Willmitzer
L
L
Fiehn
O
O
D000595:Amino Acid Sequence; D002364:Caseins / Q000032:analysis; D002853:Chromatography, Liquid; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D014357:Trypsin
D002364:Caseins; D010455:Peptides; D010750:Phosphoproteins; D014357:Trypsin