Pharmaceutical Sciences Faculty Publications

Title

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

Document Type

Article

Publication Date

6-1-2001

Journal Title

Analytical Chemistry

ISSN

0003-2700

Volume

73

Issue

11

First Page

2578

Last Page

2586

DOI

10.1021/ac010081x

PubMed ID

11403303

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 beta-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 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.

Keywords

Affinity labels, chromatography, liquid, indicators, reagents, isotopes, phosphopeptides, phosphoproteins, proteome, saccharomyces cerevisiae, spectrometry, mass, electrospray ionization

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