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 aff inity tags (PhIAT) that combines stable isotope and biotin labeling to enri ch and quantitatively measure differences in the O-phosphorylation states o f proteins. The PhIAT labeling approach involves hydroxide ion-mediated B-e limination 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 (+)-biot inyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contai ns the nucleophilic sulfhydryl and isotopic label covalently linked to a bi otin 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 -ca sein, After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-ph ase Liquid chromatography-mass spectrometry, phIAT-labeled beta -casein pep tides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified. The PhIAT labeling method was also applied t o a yeast protein extract. The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosph orylation state of proteins.