Pharmaceutical Sciences Faculty Publications
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
Recommended Citation
Goshe, Michael B.; Conrads, Thomas P.; Panisko, Ellen A.; Angell, Nicolas H.; Veenstra, Timothy D.; and Smith, Richard D., "Phosphoprotein Isotope-Coded Affinity Tag Approach for Isolating and Quantitating Phosphopeptides in Proteome-Wide Analyses" (2001). Pharmaceutical Sciences Faculty Publications. 511.
https://digitalcommons.cedarville.edu/pharmaceutical_sciences_publications/511
