TY - JOUR
T1 - Analysis of plant phosphoproteins
AU - Meimoun, P.
AU - Ambard-Bretteville, F.
AU - Colas Des Francs-Small, Catherine
AU - Valot, B.
AU - Vidal, J.
PY - 2007
Y1 - 2007
N2 - 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 P-32-phosphate-labeled proteins ill 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 I D gels and liquid chromatography/tandem mass spectrometry allowed the identification of 250 proteins belonging to various functional categories. These results validate the method for ill Vitrolin vivo studies of native/denatured individual proteins/enzymes regulated by phosphorylation and for phosphorylome studies. (C) 2007 Elsevier Inc. All rights reserved.
AB - 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 P-32-phosphate-labeled proteins ill 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 I D gels and liquid chromatography/tandem mass spectrometry allowed the identification of 250 proteins belonging to various functional categories. These results validate the method for ill Vitrolin vivo studies of native/denatured individual proteins/enzymes regulated by phosphorylation and for phosphorylome studies. (C) 2007 Elsevier Inc. All rights reserved.
U2 - 10.1016/j.ab.2007.08.022
DO - 10.1016/j.ab.2007.08.022
M3 - Article
C2 - 17910941
SN - 0003-2697
VL - 371
SP - 238
EP - 246
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -