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

Capillary liquid chromatography (muLC) interfaced to inductively coupled plasma mass spectrometry (ICPMS) is introduced as a new micromethod to determine the phosphorylation degree in phosphoproteins and phosphopeptides containing cysteine and/or methionine residues. The stoichiometric phosphorus to sulfur (31P to 32S) ratio is experimentally determined by muLC-ICPMS and converted into the degree of phosphorylation using protein/ peptide sequence information. The method is applied to the phosphoproteins beta-casein, beta-casein, and recombinant protein kinase A catalytic subunit and to synthetic phosphopeptides. The accurate data obtained by muLC-ICPMS allow quantitative assessment of the compound-specific discrimination of the electrospray ionization process between nonphosphorylated and phosphorylated proteins and peptides.