Protein phosphorylation degree: Determination by capillary liquid chromatography and inductively coupled plasma mass spectrometry

Capillary liquid chromatography (mu LC) interfaced to inductively coupled p lasma mass spectrometry (ICPMS) is introduced as a new micromethod to deter mine the phosphorylation degree in phosphoproteins and phosphopeptides cont aining cysteine and/or methionine residues. The stoichiometric phosphorus t o sulfur (P-31 to S-32) ratio is experimentally determined by mu LC-ICPMS a nd converted into the degree of phosphorylation using protein/peptide seque nce information. The method is applied to the phosphoproteins a-casein, B-c asein, and recombinant protein kinase A catalytic subunit and to synthetic phosphopeptides. The accurate data obtained by mu LC-ICPMS allow quantitati ve assessment of the compound-specific discrimination of the electrospray i onization process between nonphosphorylated and phosphorylated proteins and peptides.