Analysis of plant phosphoproteins

Anal Biochem. 2007 Dec 15;371(2):238-46. doi: 10.1016/j.ab.2007.08.022. Epub 2007 Aug 23.


Chromatography supports to purify phosphorylated proteins (P-proteins) have become available recently, yet this has not been thoroughly investigated in the case of plant materials. In this study we used a commercial affinity matrix (Qiagen) and a test plant enzyme (phosphoenolpyruvate carboxylase PEPC). The malate test and gel blot experiments probed with a specific antibody (antiphosphorylated N-terminal domain) showed that the column efficiently binds P-PEPC from Sorghum with little or no contamination by non-P-PEPC. Similar results were obtained with the low-abundance PEPC of Arabidopsis leaves when a gel filtration step (Sephadex G-200) was performed prior to the chromatography. Three-dimensional mass spectrometry analysis of immunoprecipitated PEPC in Qiagen fractions confirmed this observation. Denaturing protein extraction by cold acetone/trichloroacetic acid of fixed material led to a complete, one-step separation of P-PEPC and non-P-PEPC. At a global scale, the column captured most of the (32)P-phosphate-labeled proteins in vivo (80%), the majority of which were subsequently found in the elution fraction (88%). This was also visualized by SDS-PAGE (1D and 2D gels) followed by Pro-Q diamond staining. Analysis of the P-protein fraction by 1D gels and liquid chromatography/tandem mass spectrometry allowed the identification of 250 proteins belonging to various functional categories. These results validate the method for in vitro/in vivo studies of native/denatured individual proteins/enzymes regulated by phosphorylation and for phosphorylome studies.

Publication types

  • Validation Study

MeSH terms

  • Arabidopsis
  • Chromatography, Affinity
  • Kinetics
  • Phosphoenolpyruvate Carboxylase / isolation & purification
  • Phosphoenolpyruvate Carboxylase / metabolism
  • Phosphoproteins / analysis*
  • Phosphoproteins / isolation & purification
  • Phosphorylation
  • Plant Leaves / enzymology
  • Plant Proteins / analysis*
  • Plant Proteins / isolation & purification
  • Protein Denaturation
  • Sorghum


  • Phosphoproteins
  • Plant Proteins
  • Phosphoenolpyruvate Carboxylase