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pmid:25914713

An improved protocol to study the plant cell wall proteome.

Bruno Printz · Raphaël Dos Santos Morais · Stefanie Wienkoop · Kjell Sergeant · Stanley Lutts · Jean-Francois Hausman · Jenny Renaut

Frontiers in plant science · 2015 doi:10.3389/fpls.2015.00237

Paper

complete

Experiment

partial

Data

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Analysis

absent

Execution

absent

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Record details sci_version · id · kind · revision · content_hash · schema · created_at · updated_at · generator
id
pmid:25914713
kind
science-record
content_hash
sha256:8d7614db18566f65c2cfc8dfd1c0b49932692a792f893adbb9e68a54ae65bb25
schema
https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json
created_at
2026-11-01T15:40:08Z
updated_at
2026-11-01T18:47:22Z
generator object · 2 fields
name
corpus-assay-engine
run_id
reading-sci-2026-11-01
paper object · 10 fields
state
complete
title
An improved protocol to study the plant cell wall proteome.
doi
10.3389/fpls.2015.00237
pmid
25914713
authors array · 7 items
[0]
Bruno Printz
[1]
Raphaël Dos Santos Morais
[2]
Stefanie Wienkoop
[3]
Kjell Sergeant
[4]
Stanley Lutts
[5]
Jean-Francois Hausman
[6]
Jenny Renaut
journal
Frontiers in plant science
year
2015
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.
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Data are available via ProteomeXchange with identifier PXD001927.
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sections[0] "Abstract" paragraph 1
quote
Data are available via ProteomeXchange with identifier PXD001927.
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complete
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claim-target-result
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The hybrid three-step extraction produced the highest number of identified cell wall proteins (242 NCBInr identifiers) and the lowest proportion of non-cell wall proteins (about 30%). No inferential statistical method is reported; the source data are available through ProteomeXchange under identifier PXD001927.
kind
quantitative
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30
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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%).
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sections[0] "Abstract" paragraph 1
quote
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%).
[1] object · 3 fields
asset
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sections[0] "Abstract" paragraph 1
quote
Data are available via ProteomeXchange with identifier PXD001927.
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complete
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The hybrid three-step extraction produced the highest number of identified cell wall proteins (242 NCBInr identifiers) and the lowest proportion of non-cell wall proteins (about 30%). No inferential statistical method is reported; the source data are available through ProteomeXchange under identifier PXD001927.
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the published estimate and its evidence
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the source data with its repository provenance
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Run the published analysis
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Apply the paper's statistical method to the resolved series and compare the result with the published target.
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https://doi.org/10.3389/fpls.2015.00237
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PMC4392696
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Amount of proteins extracted by fraction and by protocol.
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PMC4392696
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18
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Results and discussion
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Table 1
caption
Amount of proteins extracted by fraction and by protocol.
rows array · 16 items
[0] array · 3 items
[0]
[1]
Protocol 1
[2]
Mass of extracted proteins (in μg/g FW)*
[1] array · 4 items
[0]
Adapted from
[1]
Watson et al., 2004
[2]
Sample 1
[3]
Sample 2
[2] array · 4 items
[0]
Extraction buffers
[1]
50 mM Na acetate, 200 mM CaCl2, pH 5.5
[2]
41.6
[3]
47.4
[3] array · 4 items
[0]
[1]
50 mM Na acetate, 3 M LiCl, pH 5.5
[2]
60.0
[3]
36.8
[4] array · 4 items
[0]
[1]
Mass of proteins isolated per g FW
[2]
101.6
[3]
84.2
[5] array · 3 items
[0]
[1]
Protocol 2
[2]
Mass of extracted proteins (in μg/g FW)*
[6] array · 4 items
[0]
Adapted from
[1]
Verdonk et al., 2012
[2]
Sample 3
[3]
Sample 4
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[0]
Extraction buffers
[1]
5 mM Na acetate, 50 mM EGTA, pH 4.6
[2]
25.5
[3]
27.6
[8] array · 4 items
[0]
[1]
5 mM Na acetate, 3 M LiCl, pH 4.6
[2]
126.0
[3]
116.8
[9] array · 4 items
[0]
[1]
Mass of proteins isolated per g FW
[2]
151.5
[3]
144.4
[10] array · 3 items
[0]
[1]
Protocol 3 - hybrid
[2]
Mass of extracted proteins (in μg/g FW)*
[11] array · 4 items
[0]
Adapted from
[1]
Feiz et al., 2006 and Verdonk et al., 2012
[2]
Sample 5
[3]
Sample 6
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[0]
Extraction buffers
[1]
5 mM Na acetate, 200 mM CaCl2, pH 4.6
[2]
156.0
[3]
171.5
[13] array · 4 items
[0]
[1]
5 mM Na acetate, 50 mM EGTA, pH 4.6
[2]
26.0
[3]
28.9
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[0]
[1]
5 mM Na acetate, 3 M LiCl, pH 4.6
[2]
50.0
[3]
50.9
[15] array · 4 items
[0]
[1]
Mass of proteins isolated per g FW
[2]
232.0
[3]
251.3
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Functional classification of the predicted cell wall proteins (CWPs) detected by LC-MS/MS analyses and based on the non-redundant Mt4.0v1 accessions.
structure object · 5 fields
pmcid
PMC4392696
section_index
19
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LC-MS/MS shotgun analysis
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11
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table object · 8 fields
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Table 2
caption
Functional classification of the predicted cell wall proteins (CWPs) detected by LC-MS/MS analyses and based on the non-redundant Mt4.0v1 accessions.
rows array · 11 items
[0] array · 3 items
[0]
Functional classification
[1]
Total (%)
[2]
% Range
[1] array · 3 items
[0]
Proteins acting on carbohydrates
[1]
26.3
[2]
24.5 – 27.8
[2] array · 3 items
[0]
Oxido-reductases
[1]
21.9
[2]
20.8 – 28.2
[3] array · 3 items
[0]
Proteins with interaction domains
[1]
14.2
[2]
10.3 – 16.7
[4] array · 3 items
[0]
Proteases
[1]
8.9
[2]
7.7 – 9.7
[5] array · 3 items
[0]
Miscellaneous proteins
[1]
8.9
[2]
5.1 – 9.9
[6] array · 3 items
[0]
Defense
[1]
7.3
[2]
5.1 – 7.7
[7] array · 3 items
[0]
Proteins related to lipid metabolism
[1]
6.9
[2]
5.7 – 11.5
[8] array · 3 items
[0]
Proteins possibly involved in signaling
[1]
2.4
[2]
2.6 – 3.1
[9] array · 3 items
[0]
Unknown function
[1]
2
[2]
0 – 2.8
[10] array · 3 items
[0]
Structural proteins
[1]
1.2
[2]
0.6 – 1.3
locator
section[19]/table-wrap[1]#T2
[3] object · 16 fields
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Information about the cell wall proteins (CWPs) detected in the fractions of the hybrid protocol by 2D-electrophoresis.
structure object · 5 fields
pmcid
PMC4392696
section_index
20
section_title
2-DE analysis
row_count
6
column_count
4
table object · 8 fields
label
Table 3
caption
Information about the cell wall proteins (CWPs) detected in the fractions of the hybrid protocol by 2D-electrophoresis.
rows array · 6 items
[0] array · 4 items
[0]
[1]
CaCl2
[2]
EGTA
[3]
LiCl
[1] array · 4 items
[0]
Number of significantly identified predicted cell wall proteins
[1]
86
[2]
76
[3]
24
[2] array · 4 items
[0]
Total number of significantly identified proteins
[1]
115
[2]
104
[3]
27
[3] array · 4 items
[0]
% of secreted proteins
[1]
74.8
[2]
73.1
[3]
88.9
[4] array · 4 items
[0]
% of fraction-specific proteins using gel electrophoresis
[1]
57.1
[2]
54.5
[3]
43.3
[5] array · 4 items
[0]
% of fraction-specific proteins using LC
[1]
24.1
[2]
22.6
[3]
35.6
locator
section[20]/table-wrap[1]#T3
[4] object · 16 fields
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section[20]/table-wrap[2]#T4
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Characterization of three groups of peroxidase identified in the LiCl fraction of the hybrid protocol.
structure object · 5 fields
pmcid
PMC4392696
section_index
20
section_title
2-DE analysis
row_count
11
column_count
4
table object · 8 fields
label
Table 4
caption
Characterization of three groups of peroxidase identified in the LiCl fraction of the hybrid protocol.
rows array · 11 items
[0] array · 4 items
[0]
Peptide mass (Da)/Peptide sequence
[1]
Group A
[2]
Group B
[3]
Group C
[1] array · 4 items
[0]
997
[1]
+
[2]
+
[3]
X
[2] array · 4 items
[0]
QLDNSFYK + Gln->pyro-Glu (N-term Q)
[1]
[2]
[3]
[3] array · 4 items
[0]
1025
[1]
+
[2]
+
[3]
+
[4] array · 4 items
[0]
QLDNSFYR + Gln->pyro-Glu (N-term Q)
[1]
[2]
[3]
[5] array · 4 items
[0]
2457
[1]
−
[2]
+
[3]
+
[6] array · 4 items
[0]
NFDRQGLDTTDLVALSGAHTIGR
[1]
[2]
[3]
[7] array · 4 items
[0]
2516
[1]
+
[2]
+
[3]
−
[8] array · 4 items
[0]
SNFDKQGLDTTDLVALSGAHTIGR
[1]
[2]
[3]
[9] array · 4 items
[0]
2591
[1]
−
[2]
−
[3]
+
[10] array · 4 items
[0]
PTLNTTYLQTLR + HexNac-(Fuc)-HexNac-Hex-(Xyl) Man(3) on N
[1]
[2]
[3]
locator
section[20]/table-wrap[2]#T4
[5] object · 16 fields
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MALDI-TOF/TOF identification of some glycosylation events detected in the fractions of the hybrid protocol.
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pmcid
PMC4392696
section_index
20
section_title
2-DE analysis
row_count
30
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table object · 8 fields
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Table 5
caption
MALDI-TOF/TOF identification of some glycosylation events detected in the fractions of the hybrid protocol.
rows array · 30 items
[0] array · 6 items
[0]
Spot
[1]
Fraction
[2]
Precursor Mass (Da)
[3]
Sequence
[4]
[5]
Remarks
[1] array · 6 items
[0]
334
[1]
CaCl2
[2]
[3]
Expansin-like A2-like
[4]
[5]
[2] array · 6 items
[0]
[1]
[2]
2375
[3]
K.VVLTDLNHNNQTDFVLSSR.A + HexNAc (N)
[4]
[5]
[3] array · 6 items
[0]
63
[1]
CaCl2
[2]
[3]
Xyloglucan-specific endoglucanase inhibitor protein
[4]
[5]
[4] array · 6 items
[0]
[1]
[2]
2915
[3]
K.ALNVSTVEPVAPFGTCFASQSISSSR.M + HexNAc (N)
[4]
[5]
[5] array · 6 items
[0]
64
[1]
CaCl2
[2]
[3]
Xyloglucan-specific endoglucanase inhibitor protein
[4]
[5]
[6] array · 6 items
[0]
[1]
[2]
2915
[3]
K.ALNVSTVEPVAPFGTCFASQSISSSR.M + HexNAc (N)
[4]
[5]
[7] array · 6 items
[0]
220
[1]
CaCl2
[2]
[3]
Peroxidase
[4]
[5]
[8] array · 6 items
[0]
[1]
[2]
1987
[3]
R.IYNETNIDTNFATLR.K + HexNAc (N)
[4]
[5]
[9] array · 6 items
[0]
[1]
[2]
2134
[3]
R.IYNETNIDTNFATLR.K + HexNAc(1)dHex(1) (N)
[4]
[5]
[10] array · 6 items
[0]
[1]
[2]
2337
[3]
R.IYNETNIDTNFATLR.K + HexNAc(2)dHex(1) (N)
[4]
[5]
[11] array · 6 items
[0]
415
[1]
CaCl2
[2]
[3]
Kunitz-type trypsin inhibitor alpha chain
[4]
[5]
[12] array · 6 items
[0]
[1]
[2]
2788
[3]
K.GGGLTVANHGENNQTCPLYVVQEK.L + HexNAc (N)
[4]
[5]
[13] array · 6 items
[0]
[1]
[2]
1545
[3]
K.HLALSDQIPSFR.V + Hex (N-term)
[4]
[5]
[14] array · 6 items
[0]
371
[1]
CaCl2
[2]
[3]
Disease resistance response protein
[4]
[5]
[15] array · 6 items
[0]
[1]
[2]
2853
[3]
FNGSTLSVLGR + HexNAc(2)Mannose(8) (N)
[4]
putative
[5]
Peptides with 1 and 2 mannose residues less are also present
[16] array · 6 items
[0]
245
[1]
CaCl2
[2]
[3]
Low homology to Polygalacturonase inhibitor
[4]
[5]
[17] array · 6 items
[0]
[1]
[2]
2947
[3]
LLPNLTGPIPQAIAR + HexNAc(2)Mannose(6) (N)
[4]
putative
[5]
[18] array · 6 items
[0]
375
[1]
CaCl2
[2]
[3]
Disease resistance response protein
[4]
[5]
[19] array · 6 items
[0]
[1]
[2]
2853
[3]
FNGSTLSVLGR + HexNAc(2)Mannose(8) (N)
[4]
putative
[5]
Peptides with 7 mannose residues are also present
[20] array · 6 items
[0]
51
[1]
CaCl2
[2]
[3]
Receptor-like protein kinase
[4]
[5]
[21] array · 6 items
[0]
[1]
[2]
2601
[3]
SVVGIQKLNVSYNR + HexNAc(2)Hex(2)Xyl(1)
[4]
putative
[5]
Further addition of HexNAc giving the peaks at 2804 and 3007
[22] array · 6 items
[0]
267
[1]
CaCl2
[2]
[3]
Glucan endo-1,3-beta-glucosidase
[4]
[5]
[23] array · 6 items
[0]
[1]
[2]
2481
[3]
K.VVVSESGWPSDGGFAATYDNTR.V + Trp →Kynurenin (W) + Hex (Kyn)
[4]
putative
[5]
[24] array · 6 items
[0]
650
[1]
EGTA
[2]
[3]
Pathogenesis-related protein 1-like
[4]
[5]
[25] array · 6 items
[0]
[1]
–
[2]
2461
[3]
R.STIISCNYDPPGNYIGQRPF.D + Hex (N-term)
[4]
[5]
[26] array · 6 items
[0]
151
[1]
EGTA
[2]
[3]
Probable pectinesterase/pectinesterase inhibitor 40-like
[4]
[5]
[27] array · 6 items
[0]
[1]
–
[2]
2737
[3]
EITNATEASQFTVR + HexNAc(Fuc)HexNAcMan(Xyl)Man(2) (N)
[4]
putative
[5]
Further addition of HexNAc giving the peaks at 2940 and 3143
[28] array · 6 items
[0]
280
[1]
LiCl
[2]
[3]
Expansin
[4]
[5]
[29] array · 6 items
[0]
[1]
[2]
2710
[3]
SLLSNNAAPAGWSFGQTYTGAQFR + Trp →Kynurenin (W) + Hex (Kyn)
[4]
putative
[5]
locator
section[20]/table-wrap[3]#T5
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[0]
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[1]
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a stem cell wall protein analysis. Following LC-MS/MS analysis the three-steps extraction procedur
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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. 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