Pharmaceutical Sciences Faculty Publications
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
Recommended Citation
Goshe, Michael B.; Veenstra, Timothy D.; Panisko, Ellen A.; Conrads, Thomas P.; Angell, Nicolas H.; and Smith, Richard D., "Phosphoprotein Isotope-Coded Affinity Tags: Application to the Enrichment and Identification of Low-Abundance Phosphoproteins" (2002). Pharmaceutical Sciences Faculty Publications. 515.
https://digitalcommons.cedarville.edu/pharmaceutical_sciences_publications/515
