An improved protocol to study the plant cell wall proteome
Cell wall proteins were extracted from alfalfa stems according to a 3-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 2 previously published methods optimized for alfalfa...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2015-04-01
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| Series: | Frontiers in Plant Science |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fpls.2015.00237/full |
| _version_ | 1811282653289119744 |
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| author | Bruno ePrintz Bruno ePrintz Raphael eDos Santos Morais Stefanie eWienkoop Kjell eSergeant Stanley eLutts Jean-Francois eHausman Jenny eRenaut |
| author_facet | Bruno ePrintz Bruno ePrintz Raphael eDos Santos Morais Stefanie eWienkoop Kjell eSergeant Stanley eLutts Jean-Francois eHausman Jenny eRenaut |
| author_sort | Bruno ePrintz |
| collection | DOAJ |
| description | Cell wall proteins were extracted from alfalfa stems according to a 3-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 2 previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the 3-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 3 protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol.This 3-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. |
| first_indexed | 2024-04-13T01:56:20Z |
| format | Article |
| id | doaj.art-bdcccf6d5a93481299bb24164c964a2b |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-13T01:56:20Z |
| publishDate | 2015-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-bdcccf6d5a93481299bb24164c964a2b2022-12-22T03:07:45ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2015-04-01610.3389/fpls.2015.00237135912An improved protocol to study the plant cell wall proteomeBruno ePrintz0Bruno ePrintz1Raphael eDos Santos Morais2Stefanie eWienkoop3Kjell eSergeant4Stanley eLutts5Jean-Francois eHausman6Jenny eRenaut7Luxembourg Institute of Science and TechnologyUniversité catholique de LouvainLuxembourg Institute of Science and TechnologyUniversity of ViennaLuxembourg Institute of Science and TechnologyUniversité catholique de LouvainLuxembourg Institute of Science and TechnologyLuxembourg Institute of Science and TechnologyCell wall proteins were extracted from alfalfa stems according to a 3-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 2 previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the 3-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 3 protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol.This 3-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.http://journal.frontiersin.org/Journal/10.3389/fpls.2015.00237/fullGlycosylationPlant ProteinsProteomicsEGTAcell wall proteins |
| spellingShingle | Bruno ePrintz Bruno ePrintz Raphael eDos Santos Morais Stefanie eWienkoop Kjell eSergeant Stanley eLutts Jean-Francois eHausman Jenny eRenaut An improved protocol to study the plant cell wall proteome Frontiers in Plant Science Glycosylation Plant Proteins Proteomics EGTA cell wall proteins |
| title | An improved protocol to study the plant cell wall proteome |
| title_full | An improved protocol to study the plant cell wall proteome |
| title_fullStr | An improved protocol to study the plant cell wall proteome |
| title_full_unstemmed | An improved protocol to study the plant cell wall proteome |
| title_short | An improved protocol to study the plant cell wall proteome |
| title_sort | improved protocol to study the plant cell wall proteome |
| topic | Glycosylation Plant Proteins Proteomics EGTA cell wall proteins |
| url | http://journal.frontiersin.org/Journal/10.3389/fpls.2015.00237/full |
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