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. 2015 Apr 10;6:237.
doi: 10.3389/fpls.2015.00237. eCollection 2015.

An Improved Protocol to Study the Plant Cell Wall Proteome

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Free PMC article

An Improved Protocol to Study the Plant Cell Wall Proteome

Bruno Printz et al. Front Plant Sci. .
Free PMC article

Abstract

Cell wall proteins were extracted from alfalfa stems according to a three-steps extraction procedure using sequentially CaCl2, EGTA, and LiCl-complemented buffers. The efficiency of this protocol for extracting cell wall proteins was compared with the two previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the three-steps extraction procedure resulted in the identification of the highest number of cell wall proteins (242 NCBInr identifiers) and gave the lowest percentage of non-cell wall proteins (about 30%). However, the three protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol. This three-step protocol was therefore selected for a more detailed proteomic characterization using 2D-gel electrophoresis. With this technique, 75% of the identified proteins were shown to be fraction-specific and 72.7% were predicted as belonging to the cell wall compartment. Although, being less sensitive than LC-MS/MS approaches in detecting and identifying low-abundant proteins, gel-based approaches are valuable tools for the differentiation and relative quantification of protein isoforms and/or modified proteins. In particular isoforms, having variations in their amino-acid sequence and/or carrying different N-linked glycan chains were detected and characterized. This study highlights how the extracting protocols as well as the analytical techniques devoted to the study of the plant cell wall proteome are complementary and how they may be combined to elucidate the dynamism of the plant cell wall proteome in biological studies. Data are available via ProteomeXchange with identifier PXD001927.

Keywords: EGTA; cell wall; glycosylation; plant; proteomics.

Figures

Figure 1
Figure 1
Diagram overview of the cell wall protein extraction protocols. Protocol 1, adapted from Watson et al. (2004); Protocol 2, adapted from Verdonk et al. (2012); Protocol 3 (hybrid), adapted from Verdonk et al. (2012) and Feiz et al. (2006).
Figure 2
Figure 2
SDS-PAGE protein profiles of the different fractions extracted with the tested protocols. (A) protocol 1, adapted from Watson et al. (2004) (B) protocol 2, adapted from Verdonk et al. (2012) (C) protocol 3 (hybrid), adapted from Verdonk et al. (2012) and Feiz et al. (2006): each lane was loaded with 20 μg of proteins on Criterion XT precast 1D gel 12% Bis-Tris (Bio-Rad). The gels were stained with Coomassie blue (InstantBlue, Expedeon).
Figure 3
Figure 3
Venn diagrams showing the repartition of the cell wall proteins (CWPs) identified by LC-MS/MS analysis according to (A) the NCBInr accessions and (B) the non-redundant Mt4.0v1 accessions obtained after blasting the NCBInr accessions on the Medicago truncatula genome.
Figure 4
Figure 4
Functional classification of the cell wall proteins (CWPs) identified by LC-MS/MS analysis of the different fractions. Calculations were made relatively to the number of non-redundant Mt4.0v1 accessions identified in each fraction. A red line was arbitrary drown at the level 20% to allow a rapid comparison of the most represented classes of proteins in each fraction. (A) protocol 1, adapted from Watson et al. (2004) (B) protocol 2, adapted from Verdonk et al. (2012) (C) protocol 3 (hybrid), adapted from Verdonk et al. (2012) and Feiz et al. (2006). Note the differences observed in the CaCl2 fraction of the protocol adapted from Watson et al. (2004) (A) and the CaCl2 fraction of the “hybrid” protocol (C). These differences are related with the 2 different procedures of cell wall isolation, the first based on filtration on Büchner devices, the second being based on washes in various sucrose concentrations.
Figure 5
Figure 5
2D-Electrophoresis of the proteins extracted in each fraction of the hybrid protocol. Proteins were separated on Immobiline DryStrip 3-10 NL, 24 cm (GE Healthcare) and further migrated on 2D-HPE Large-Gels NF 12.5% (Serva Electrophoresis GmbH). Proteins were post-stained with LavaPurple (Serva Electrophoresis GmbH).
Figure 6
Figure 6
Venn diagram showing the repartition of the 194 proteins identified by MALDI-TOF-TOF. Numbers in black correspond to the number of plant proteins, numbers in red indicate the number of fungal proteins. In italics, the percentage of cell wall proteins (CWPs) is indicated.
Figure 7
Figure 7
Localization of some protein isoforms detected after spot detection, picking, digestion, and identification. In the LiCl fraction, three groups of spots containing peroxidase were identified and arbitrary named group A, B, and C for further MALDI-TOF/TOF characterization.

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