Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls.
BACKGROUND
The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Reaching such a goal closely depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific difficulties: (i) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure, (ii) polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50%) of predicted intracellular proteins. Since isolated cell walls should hold secreted proteins, one can imagine alternative procedures to prepare cell walls containing a lower proportion of contaminant proteins.


RESULTS
The rationales of several published procedures to isolate cell walls for proteomics were analyzed, with regard to the bioinformatic-predicted subcellular localization of the identified proteins. Critical steps were revealed: (i) homogenization in low ionic strength acid buffer to retain CWP, (ii) purification through increasing density cushions, (iii) extensive washes with a low ionic strength acid buffer to retain CWP while removing as many cytosolic proteins as possible, and (iv) absence of detergents. A new procedure was developed to prepare cell walls from etiolated hypocotyls of Arabidopsis thaliana. After salt extraction, a high proportion of proteins predicted to be secreted was released (73%), belonging to the same functional classes as proteins identified using previously described protocols. Finally, removal of intracellular proteins was obtained using detergents, but their amount represented less than 3% in mass of the total protein extract, based on protein quantification.


CONCLUSIONS
The new cell wall preparation described in this paper gives the lowest proportion of proteins predicted to be intracellular when compared to available protocols. The application of its principles should lead to a more realistic view of the cell wall proteome, at least for the weakly bound CWP extractable by salts. In addition, it offers a clean cell wall preparation for subsequent extraction of strongly bound CWP.
Feiz
Leila
L
Surfaces Cellulaires et Signalisation chez les Végétaux, UMR 5546 CNRS-Université Paul Sabatier-Toulouse III, Pôle de Biotechnologie végétale, 24, Chemin de Borde Rouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France.|Department of Plant Science and Plant Pathology, Agriculture and Biological Sciences Faculty, Montana State University-Bozeman, Bozeman, MT 59717-3150, USA.
Irshad
Muhammad
M
Surfaces Cellulaires et Signalisation chez les Végétaux, UMR 5546 CNRS-Université Paul Sabatier-Toulouse III, Pôle de Biotechnologie végétale, 24, Chemin de Borde Rouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France.
Pont-Lezica
Rafael F
RF
Surfaces Cellulaires et Signalisation chez les Végétaux, UMR 5546 CNRS-Université Paul Sabatier-Toulouse III, Pôle de Biotechnologie végétale, 24, Chemin de Borde Rouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France.
Canut
Hervé
H
Surfaces Cellulaires et Signalisation chez les Végétaux, UMR 5546 CNRS-Université Paul Sabatier-Toulouse III, Pôle de Biotechnologie végétale, 24, Chemin de Borde Rouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France.
Jamet
Elisabeth
E
Surfaces Cellulaires et Signalisation chez les Végétaux, UMR 5546 CNRS-Université Paul Sabatier-Toulouse III, Pôle de Biotechnologie végétale, 24, Chemin de Borde Rouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France.