Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome

Nat Biotechnol. 2001 Apr;19(4):379-82. doi: 10.1038/86783.


The current progression from genomics to proteomics is fueled by the realization that many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence. Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry after two-dimensional electrophoretic separation, it can be difficult to elucidate low-abundance proteins of interest in the presence of a large excess of relatively abundant proteins. Therefore, for effective proteome analysis it becomes critical to enrich the sample to be analyzed in subfractions of interest. For example, the analysis of protein kinase substrates can be greatly enhanced by enriching the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies, the enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Here, we describe a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts, and for subsequently identifying the phosphoproteins and sites of phosphorylation. The method, which involves chemical replacement of the phosphate moieties by affinity tags, should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biochemistry / methods*
  • Biotinylation
  • Caseins / chemistry
  • Cattle
  • Chick Embryo
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Gas Chromatography-Mass Spectrometry
  • Models, Chemical
  • Molecular Sequence Data
  • Ovalbumin / chemistry
  • Phosphorylation
  • Phosphoserine / chemistry
  • Proteins / analysis*
  • Proteins / chemistry*
  • Signal Transduction
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
  • Threonine / chemistry


  • Caseins
  • Proteins
  • Phosphoserine
  • Threonine
  • Ovalbumin