Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls.

Leila Feiz,Elisabeth Jamet,Muhammad Irshad,Rafael F Pont-Lezica,Hervé Canut

doi:10.1186/1746-4811-2-10

Abstract

BackgroundThe 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.ResultsThe 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.ConclusionThe 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.

Highlights

The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments
The aim of the present study is to present a comparative analysis of different methods previously published to prepare cell walls for proteomic studies
The results indicate that such a method significantly reduces the number of proteins without predicted signal peptide

Summary

Introduction

The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Cell walls are natural composite structures, mostly made of high molecular weight polysaccharides, proteins, and lignins, the latter found only in specific cell types They are dynamic structures contributing to the general morphology of the plant. Cell wall proteins (CWP) represent a minor fraction of the wall mass: 5–10% in primary cell walls of dicots, as reported for cell suspension cultures, but accurate determinations in various plant organs are still lacking [6]. Despite their low abundance, CWP contribute, at least in part, to the dynamic of cell walls. CWP can be involved in modification of cell wall components, wall structure, signalling, and interactions with plasma membrane proteins at the cell surface [7]

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