Prev. Article Next Article Table of Contents

Article

Phosphoprotein Isotope-Coded Affinity Tag Approach for Isolating and Quantitating Phosphopeptides in Proteome-Wide Analyses

Michael B. Goshe, Thomas P. Conrads, Ellen A. Panisko, Nicolas H. Angell, Timothy D. Veenstra, and Richard D. Smith*

Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, MSIN: K8-98, Richland, Washington 99352

Anal. Chem., 2001, 73 (11), pp 2578–2586

DOI: 10.1021/ac010081x

Publication Date (Web): April 28, 2001

In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.

Abstract

A 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 β-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D₀) or four alkyl deuteriums (EDT-D₄) followed by biotinylation of the EDT-D₀/D₄ moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was 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 β-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 β-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.

View: Full Text HTML | Hi-Res PDF