Analysis of plant phosphoproteins

P. Meimoun, F. Ambard-Bretteville, Catherine Colas Des Francs-Small, B. Valot, J. Vidal

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)238-246
JournalAnalytical Biochemistry
Volume371
Issue number2
DOIs
Publication statusPublished - 2007

Fingerprint

Dive into the research topics of 'Analysis of plant phosphoproteins'. Together they form a unique fingerprint.

Cite this