Analysis of plant phosphoproteins.
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.
Meimoun
P
P
Institut de Biotechnologie des Plantes, CNRS-UMR 8618, Bâtiment 630, Université de Paris-Sud, 91405 Orsay Cedex, France.
Ambard-Bretteville
F
F
Colas-des Francs-Small
C
C
Valot
B
B
Vidal
J
J
D017360:Arabidopsis; D002846:Chromatography, Affinity; D007700:Kinetics; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000302:isolation & purification; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D011489:Protein Denaturation; D045868:Sorghum
D010750:Phosphoproteins; D010940:Plant Proteins; D010730:Phosphoenolpyruvate Carboxylase