Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures

Anal Chem. 2003 Oct 15;75(20):5441-50. doi: 10.1021/ac0342774.


Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Carbon Isotopes / chemistry
  • Caseins / analysis
  • Caseins / metabolism
  • Cell Extracts / chemistry
  • Cell Line, Tumor
  • Complex Mixtures / chemistry*
  • Databases, Protein
  • Gas Chromatography-Mass Spectrometry / methods
  • Glass / chemistry
  • Humans
  • Isotope Labeling / methods*
  • Molecular Sequence Data
  • Molecular Weight
  • Nitrogen Isotopes / chemistry
  • Peptide Fragments / analysis
  • Phosphopeptides / analysis*
  • Phosphoproteins / analysis
  • Phosphoproteins / metabolism
  • Phosphoserine / chemistry
  • Phosphothreonine / chemistry
  • Photochemistry
  • Trypsin / chemistry
  • Trypsin / metabolism


  • Carbon Isotopes
  • Caseins
  • Cell Extracts
  • Complex Mixtures
  • Nitrogen Isotopes
  • Peptide Fragments
  • Phosphopeptides
  • Phosphoproteins
  • Phosphothreonine
  • Phosphoserine
  • Trypsin