Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls
- Leila Feiz†1, 2,
- Muhammad Irshad†1,
- Rafael F Pont-Lezica1,
- Hervé Canut1 and
- Elisabeth Jamet1Email author
© Feiz et al; licensee BioMed Central Ltd. 2006
Received: 27 February 2006
Accepted: 27 May 2006
Published: 27 May 2006
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.
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.
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.
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 walls are involved in cell expansion and division, and they are sources of signals for molecular recognition within the same or between different organisms [1–5]. 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 . 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 .
Proteomics appears to be a suitable method to identify a large number of CWP thus providing information for many genes still lacking a function. Recent publications on cell wall proteomics have shown that more than 50% of the identified proteins were known to be intracellular proteins in higher plants [8, 9], green alga  and fungi . Different techniques unrelated to proteomics, such as biotinylation of cell surface proteins, or immunoelectron microscopy, also suggested a cell wall location for some glycolytic enzymes, proposing that they are bona fide components of the yeast cell wall . However, the reliability of protein profiling for a compartment like the cell wall, strongly depends on the quality of the preparation. Unfortunately, the classical methods to check the purity of a particular fraction are not conclusive for proteomic studies, since the sensibility of the analysis by mass spectrometry is 10 to 1000 times more sensitive than enzymatic or immunological tests using specific markers. Our experience in the field has shown that the most efficient way to evaluate the quality of a cell wall preparation is (i) to identify all the proteins extracted from the cell wall by mass spectrometry, and (ii) to perform extensive bioinformatic analysis to determine if the identified proteins contain a signal peptide, and no retention signals for other cell compartments [12–15]. It is then possible to conclude about the quality of the cell wall preparation by calculating the proportion of predicted secreted proteins to intracellular ones.
The aim of the present study is to present a comparative analysis of different methods previously published to prepare cell walls for proteomic studies. These methods will be evaluated by the proportion of proteins predicted to be secreted after bioinformatic analysis as stated above. A new method is presented, based on classical cell wall preparations, but adapted to the new technologies. The results indicate that such a method significantly reduces the number of proteins without predicted signal peptide.
Results and discussion
Several strategies have been designed to gain access to CWP. The most labile CWP, i.e. those having little or no interactions with cell wall components, can be recovered in culture media of cell suspension cultures  or liquid cultured seedlings . Extracellular fluids can be harvested from cell suspension cultures [12, 16] or intact organs such as leaves . However, such analysis cannot be done in all cases. It is then necessary to isolate cell walls starting with a drastic mechanical disruption of the material of interest. Consequently, labile CWP may be lost, and intracellular proteins or organelle fragments may contaminate cell wall preparations.
To design a procedure for cell wall isolation and subsequent protein extraction, several general features should be kept in mind. Plant and fungal cell walls are mainly built up with highly dense polysaccharides. This property can be used to purify them through density gradients by centrifugation. The biochemical structure of walls is complex, and CWP can be bound to the matrix by Van der Waals forces, hydrogen bonds, and hydrophobic or ionic attractions. Such interactions can also be modulated by the composition of the isolation medium. Commonly, a low ionic strength is preferred to preserve ionic bonds, but also to dilute the ionic strength of the cell wall itself. An acidic pH is chosen to maintain the interactions between proteins and polysaccharides as in planta. Once isolated, cell walls are classically treated by CaCl2 buffers to release proteins, and by LiCl buffers for extraction of glycoprotein [17, 18]. The use of detergents has also been reported to extract proteins strongly embedded in the polysaccharide matrix, like wall associated kinases . Finally, CWP can be covalently bound to cell wall components so that they are resistant to salt-extraction. At present, there is no satisfactory procedure to isolate them. We analyze recent publications using different methods to isolate cell walls from plants or yeast prior to proteomic analysis [8, 9, 20].
Analysis of early methods
Altogether, this evaluation of three procedures to isolate cell walls from plants or fungi prior to proteomic analyses shows that they all produce a material containing a high proportion of proteins predicted to be intracellular, suggesting they are contaminants. Even if a careful bioinformatic analysis allows the discrimination between secreted and intracellular proteins, the time and effort consumed is not satisfactory.
A modified method to prepare plant cell walls
From the analysis of the presented methods and other classical cell wall preparations used for the purification of cell wall enzymes [23, 24], several points appear to be essential for the purification of cell walls. First, the presence of NaCl at early steps of cell wall preparations of M. sativa and C. albicans in grinding or washing buffers might induce a release of CWP even at a low concentration . This might indirectly increase the proportion of intracellular proteins sticking non-specifically to cell wall polysaccharides. The use of a low ionic strength buffer for tissue grinding and subsequent washes to purify cell walls appears as an interesting alternative to prevent loss of CWP. Second, the protocol used for A. thaliana cell suspension cultures is the only one including a purification of cell walls through a dense medium, i.e. sedimentation in 10% glycerol. Proteins predicted to be secreted represented 50% of the identified proteins. It seems that a series of sedimentations/centrifugations in solutions of increasing densities would help in eliminating organelles and other vesicles less dense than cell wall polysaccharides [23, 24]. Third, despite repeated washes, the cell wall preparation from A. thaliana cell suspension cultures still contained a high proportion of proteins predicted to be intracellular. A way to eliminate soluble contaminants such as intracellular proteins is to perform extensive washes of cell walls with a low ionic strength buffer . Finally, the addition of polyvinyl polypyrrolidone (PVPP) to trap plant phenolic compounds  as well as anti-proteases to limit protein degradation during the manipulations , improves the quality of protein identification by mass spectrometry. Our procedure was established on the basis of these requirements.
Sequential salt extraction of proteins from cell walls
A recent review on 281 CWP identified in proteomic studies by mass spectrometry  concluded that more than 60% of them have a basic pI, and around 80% in salt-extractable fractions. This is a serious problem for the separation of CWP by the classical 2D-GE, since it is well known that basic glycoproteins are poorly resolved by this technique . We have then used 1D-GE for the separation, each band stained with Coomassie™ blue was digested by trypsin and further analyzed by MALDI-TOF MS or LC/MS/MS. Each protein was analyzed using several bioinformatic programs as described above. Seventy-three different proteins predicted to be secreted were identified in this study, whereas only 12 proteins predicted to be intracellular and 11 proteins predicted to have trans-membrane domains were found.
The protocol of CWP extraction we used is almost the same as the one employed with M. sativa stems . But comparison of Figures 2B and 5B shows big differences in the proportion of proteins predicted to be intracellular or having trans-membrane domains (50% for M. sativa vs 27% for A. thaliana). Since the main difference between the two protocols is the addition of centrifugations through a dense medium, it shows that this step is critical for the purity of cell walls.
The proteins predicted to be secreted identified in this study belong to the same functional classes as those described in previous cell wall proteomes established from cell wall preparations [8, 9, 25]. Shortly, these functional classes comprise proteins acting on polysaccharides (e.g. glycoside hydrolases, carbohydrate esterases, expansins), proteases, proteins with interacting domains (e.g. lectins, leucine-rich repeat proteins, enzyme inhibitors), oxido-reductases (e.g. peroxidases, berberine-bridge enzymes), proteins involved in signaling processes (e.g. arabinogalactan proteins), structural proteins, proteins of unknown function and miscellaneous proteins . The new protocol appears to be more efficient since a large proportion of identified proteins are predicted to be targeted to the compartment of interest.
The new cell wall preparation procedure followed by salt extraction of proteins described in this paper gives the lowest proportion of proteins predicted to be intracellular when compared with other available protocols and allows the identification of proteins fitting in the same functional classes. Addition of a step including detergent treatment revealed the presence of minor amounts of a few additional proteins predicted to be secreted, but of many proteins predicted to be intracellular. Prediction of the sub-cellular localization of proteins by different bioinformatic programs appeared as an essential tool to evaluate cell wall purification procedures. However, it should not be considered satisfactory to determine the sub-cellular localization of any protein identified by a proteomic analysis. Additional experiments performed in planta, such as immunolocalization or localization of fluorescent protein fusions, are required to confirm it. The application of the principles of cell wall purification described in this paper 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.
Plant material and isolation of cell walls
One hundred and fifty mg of A. thaliana seeds (ecotype Columbia 0) were sowed per Magenta box containing Murashige and Skoog medium  supplemented with 2% w/v sucrose and 1.2% w/v agar. Seedlings were grown at 23°C in the dark for 11 days. For one experiment, hypocotyls from 16 Magenta boxes were collected. One cm long hypocotyls were cut below the cotyledons and above the root, washed with distilled water and transferred into 500 mL of 5 mM acetate buffer, pH 4.6, 0.4 M sucrose and protease inhibitor cocktail (Sigma) 1 mL per 30 g of hypocotyl fresh weight. The mixture was ground in a blender at full speed for 15 min (Figure 4). After adding PVPP (1 g per 10 g fresh weight of hypocotyls), the mixture was incubated in cold room for 30 min while stirring. Cell walls were separated from soluble cytoplasmic fluid by centrifugation of the homogenate for 15 min at 1000 × g and 4°C. The pellet (CW1 in Figure 4) was further purified by two successive centrifugations in 500 mL of 5 mM acetate buffer, pH 4.6, respectively 0.6 M and 1 M sucrose. The residue (CW3) was washed with 3 L of 5 mM acetate buffer, pH 4.6, on a nylon net (25 μm pore size). The resulting cell wall fraction (CW4) was ground in liquid nitrogen in a mortar with a pestle prior to lyophilization. Starting with 16 g fresh weight of hypocotyls, this process resulted in 1.3 g dry powder.
Sequential proteins extraction and identification
Typically, 0.65 g of lyophilized cell walls was used for one experiment. Proteins were extracted by successive salt solutions in this order: two extractions each time with 6 mL CaCl2 solution (5 mM acetate buffer, pH 4.6, 0.2 M CaCl2 and 10 μL protease inhibitor cocktail), followed by two extractions with 6 mL LiCl solution (5 mM acetate buffer, pH 4.6, 2 M LiCl and 10 μL protease inhibitor cocktail). Cell walls were resuspended by vortexing for 5–10 min at room temperature, and then centrifuged for 15 min at 4000 × g and 4°C. Supernatants were desalted using Econo-Pac® 10 DG columns (Bio-Rad) equilibrated with 0.2 formic acid ammonium salt. The extract were lyophilized and resuspended in sample buffer for separation of proteins by 1D-GE, as previously described .
The next extraction was carried out by SDS and DTT. The cell wall preparation was treated with 12 mL solution containing 62.5 mM Tris, 4% SDS, 50 mM DTT, pH 6.8 (HCl). The mixture was boiled for 5 min and centrifuged for 15 min at 40000 × g and 4°C. The supernatant was dialyzed against 1 L H2O in Spectra/Por® membrane 10 kDa MWCO bags (Spectrum Medical Industries) at room temperature, then concentrated by successive centrifugation using the Centriprep® centrifugal filter devices (YM-10 kDa membrane) (Millipore) at 4000 × g followed by speed vacuum centrifugation.
The protein content of each extract was measured using the Bradford method  with the Coomassie™ protein assay reagent kit (Pierce) using bovine serum albumin (BSA) as standard.
The sequences of the identified proteins were subsequently analyzed with several bioinformatic programs to predict their sub-cellular localization [29–31]. In some cases, predictions were not the same with the three programs. Results are then indicated as "not clear". Data are described in Tables 1–5 (additional data).
one or two dimensional gel electrophoresis
cell wall proteins
3- [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
ethylene diamino tetraacetic acid
liquid chromatography-tandem mass spectrometry
matrix-assisted laser desorption ionization-time of flight
sodium dodecyl sulphate
The authors are grateful to the Université Paul Sabatier (Toulouse, France) and the CNRS (France) for support. M.I. is a fellow from the Higher Education Commission, Islamabad, Pakistan and from the French government on the behalf of SFERE. Mass spectrometry analyses were performed on the Plate-Forme de Spectrométrie de Masse in Toulouse, France.
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