Phosphoproteomics, nowadays, represents a front line in functional proteomics as testified by the number of papers recently appearing in the literature. In an attempt to improve and simplify the methods so far suggested we have set up a simple isotope-coded approach to label and quantitate phospho-Ser/-Thr residues in protein mixtures. First of all, after appropriate oxidation of cysteine/cystine residues followed by tryptic hydrolysis, we have optimised and simplified theβ-elimination reaction to get the corresponding alkene moiety from the phosphate esters. This was achieved by (a) separating the elimination reaction from the addition reaction, (b) the use of Ba(OH)₂ as alkali reagent and (c) its further elimination by the simple addition of solid CO₂ to the peptide mixture. The Michael reaction was then performed, after the removal of BaCO₃ by centrifugation, by adding dithiothreitol (DTT) to the peptide mixture. Finally, the direct purification of the modified phosphopeptides was performed on a thiol-sepharose column. The availability of fully deuterated DTT, introducing a 6 Da difference with respect to the non-deuterated species, allows quantitation of the differential extent of signalling modification when analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and liquid chromatography/mass spectrometry. The entire procedure has been set up by using bovine α-casein, and resulted in the identification of all the phosphorylated tryptic peptides, including the tetraphosphorylated peptides, which escaped all previously reported procedures

Keywords: alpha casein, phosphoproteome, ICAT, mass spectrometry