Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin-layer cellulose plates

Electrophoresis. Mar-Apr 1994;15(3-4):544-54. doi: 10.1002/elps.1150150173.


Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the analysis of the phosphoamino acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Autoradiography / methods
  • Chromatography, Thin Layer / methods
  • Electrophoresis, Gel, Two-Dimensional / methods
  • Electrophoresis, Polyacrylamide Gel / methods*
  • Endopeptidases / metabolism
  • Humans
  • Indicators and Reagents
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Peptide Mapping / methods*
  • Phosphates / metabolism
  • Phosphopeptides / analysis*
  • Phosphopeptides / chemistry
  • Phosphopeptides / isolation & purification
  • Phosphorus Radioisotopes
  • Protein Biosynthesis
  • Proteins / isolation & purification*
  • Substrate Specificity


  • Indicators and Reagents
  • Phosphates
  • Phosphopeptides
  • Phosphorus Radioisotopes
  • Proteins
  • Endopeptidases