Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses

ii method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins. The PhIAT labeling approach involves hydroxide ion-mediated B-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D-0) or four alkyl deuteriums (EDT-D-4) followed by biotinylation of the EDT-D-0/D-4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, nas synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process. The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta -casein, After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-phase Liquid chromatography-mass spectrometry, phIAT-labeled beta -casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified. The PhIAT labeling method was also applied to a yeast protein extract. The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins.