Pharmaceutical Sciences Faculty Publications

Title

Phosphoprotein Isotope-Coded Affinity Tags: Application to the Enrichment and Identification of Low-Abundance Phosphoproteins

Document Type

Article

Publication Date

2-1-2002

Journal Title

Analytical Chemistry

ISSN

0003-2700

Volume

74

Issue

3

First Page

607

Last Page

616

DOI

10.1021/ac015528g

PubMed ID

11838682

Abstract

The use of a phosphoprotein isotope-coded affinity tag (PhIAT), which employs differential isotopic labeling and biotinylation, has been shown capable of enriching and identifying mixtures of low-abundance phosphopeptides. A denatured solution of beta-casein was labeled using the PhIAT method, and after proteolytic digestion, the labeled peptides were isolated using immobilized 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 beta-casein were identified along with the phosphorylated peptides from alphas1-casein and alphas2-casein, known low-level (<5%) contaminants of commercially available beta-casein. All of the casein-phosphorylated residues identified by the present PhIAT approach correspond to previously documented sites of phosphorylation. The results illustrate the efficacy of the PhIAT-labeling strategy to not only enrich mixtures for phosphopeptides but also, more importantly, permit the detection and identification of low-level phosphopeptides. In addition, the differences in the phosphorylation state could be determined between phosphopeptides in comparative samples by stoichiometric conversion using the light and heavy isotopic versions of the PhIAT reagents. Overall, our results exemplify the application of the PhIAT approach and demonstrate its utility for proteome-wide phosphoprotein identification and quantitation.

Keywords

Affinity labels, caseins, chromatography, liquid, isotopes, mass spectrometry, peptide mapping, phosphoproteins

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