Phosphoproteins analysis in plants: a proteomic approach.
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.
Laugesen
Sabrina
S
Laboratoire de Protéomique, UR 1199 INRA, 2 Place Viala, 34060 Montpellier cedex 1, France.
Messinese
Elsa
E
Hem
Sonia
S
Pichereaux
Carole
C
Grat
Sabine
S
Ranjeva
Raoul
R
Rossignol
Michel
M
Bono
Jean-Jacques
JJ
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
D010750:Phosphoproteins; D010940:Plant Proteins