title: Phosphoproteins analysis in plants: a proteomic approach.
abstract: The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants.
authors.affiliation: Laboratoire de Protéomique, UR 1199 INRA, 2 Place Viala, 34060 Montpellier cedex 1, France.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Laugesen
authors.forename: Elsa
authors.initials: E
authors.lastname: Messinese
authors.forename: Sonia
authors.initials: S
authors.lastname: Hem
authors.forename: Carole
authors.initials: C
authors.lastname: Pichereaux
authors.forename: Sabine
authors.initials: S
authors.lastname: Grat
authors.forename: Raoul
authors.initials: R
authors.lastname: Ranjeva
authors.forename: Michel
authors.initials: M
authors.lastname: Rossignol
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Bono
mesh_terms: D017360:Arabidopsis / Q000737:chemistry*; D005591:Chemical Fractionation; D002845:Chromatography; D015180:Electrophoresis, Gel, Two-Dimensional; D046913:Medicago truncatula / Q000737:chemistry*; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010750:Phosphoproteins; D010940:Plant Proteins