Phosphoprotein isotope-coded affinity tags: Application to the enrichment and identification of low-abundance phosphoproteins

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

149 Scopus citations


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 β-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 β-casein were identified along with the phosphorylated peptides from αS1-casein and αS2-casein, known low-level (<5%) contaminants of commercially available β-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.

Original languageEnglish
Pages (from-to)607-616
Number of pages10
JournalAnalytical Chemistry
Issue number3
StatePublished - 1 Feb 2002
Externally publishedYes


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