A novel approach using different isotopic labeling and biotinylation has been developed for the enrichment and quantitation of phosphoseryl and phosphothreonyl-peptides. The phosphoprotein isotope-coded affinity tag (PhIAT) exploits the high affinity biotin-avidin interaction to isolate modified phosphopeptides from a complex mixture of peptides. The PhIAT strategy for quantifying and enriching mixtures for phosphopeptides was demonstrated using a commercially available sample of the phosphoprotein B-casein. A denatured solution of B-casein was labeled using the PhIAT method and after proteolytic digestion, the labeled peptides were isolated using immobilize avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from B-casein were identified as were phophorylated peptides from as1-casein and ase-casein, known low-level (
Revised: January 17, 2003 |
Published: February 1, 2002
Citation
Goshe M., T.D. Veenstra, E.A. Panisko, T.P. Conrads, N.H. Angell, and R.D. Smith. 2002.Phosphoprotein Isotope-coded Affinity Tags: Application to the Enrichment and Identification of Low-Abundance Phosphoproteins.Analytical Chemistry 74, no. 3:607-616. PNWD-SA-5412.