Phosphoprotein Isotope-coded Affinity Tags: Application to the Enrichment and Identification of Low-Abundance Phosphoproteins
- Case Western reserve University
- BATTELLE (PACIFIC NW LAB)
- ASSOC WESTERN UNIVERSITY
- Imperial College of Science
A novel approach using different isotopic labeling and biotinylation has been developed for the enrichment and quantitation of phosphoseryl and phosphothreonyl-peptides. The phosphoprotein isotope-coded affinity tag (PhIAT) exploits the high affinity biotin-avidin interaction to isolate modified phosphopeptides from a complex mixture of peptides. The PhIAT strategy for quantifying and enriching mixtures for phosphopeptides was demonstrated using a commercially available sample of the phosphoprotein B-casein. A denatured solution of B-casein was labeled using the PhIAT method and after proteolytic digestion, the labeled peptides were isolated using immobilize 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 B-casein were identified as were phosphorylated peptides from as1-casein and ase-casein, known low-level (< 5%) contaminants of commercially available B-casein. All of the identified phosphopeptides from these caseins have been previously documented to be phosphorylated at the sites elucidated by the PhIAT approach. The results illustrate the efficancy of the PhIAT-labeling strategy to enrich mixtures for phosphopeptides and permit the detection and identification of low abundance phosphopeptides. In addition, experiments using light and heavy isotopic version of the PhIAT reagents demonstrated that a 10% difference in phosphorylation state could be determined between phosphopeptides in comparative samples.
- Research Organization:
- Pacific Northwest National Lab., Richland, WA (US)
- Sponsoring Organization:
- US Department of Energy (US)
- DOE Contract Number:
- AC06-76RL01830
- OSTI ID:
- 15002838
- Report Number(s):
- PNWD-SA-5412
- Journal Information:
- Analytical Chemistry, Journal Name: Analytical Chemistry Journal Issue: 3 Vol. 74
- Country of Publication:
- United States
- Language:
- English
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