content contamination. The selection of an appropriate extraction
protocol is a crucial step in a proteomic approach, and subsequent
compatibility with the analytical techniques (e.g., mass spectrometry) has to be considered.
The most commonly used technique for plant APF extraction is
the vacuum infiltration-centrifugation (VIC), described by Klement in 1965 [6]. The VIC involves two critical steps: vacuum
infiltration with appropriate extraction buffer and centrifugation
[7]. The composition of the infiltration buffer is one of the key
features of this method, as it should combine the maintenance of
osmotic pressure to prevent plasma membrane damage and cytoplasm content leaking, and the efficiency to extract cell wall proteins
[7]. High concentrations of ionic reagents, such as KCl, NaCl, or
LiCl, are normally considered to guarantee weakly bound cell wall
protein solubilization [2]. The centrifugation step is also limiting,
as centrifugation speed may cause damage to cell walls and
membranes [7].
Here, we present a vacuum protocol optimized for woody plant
leaves, namely Vitis vinifera and Coffea arabica. This protocol is
relatively easy to perform, allows obtaining an acceptable protein
yield, avoids contamination with cytosolic components, and is
compatible with high-throughput mass spectrometry-based proteomic approaches, namely shotgun proteomics. We discuss critical
points within the extraction procedure, namely the influence of the
composition of the infiltration buffer. Using this protocol, we have
isolated apoplastic proteins from both coffee and grapevine leaves
[8–10], yielding a good coverage of the apoplast proteome.
2 Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water, to attain a sensitivity of 18 MΩ cm at 25
C) and
analytical-grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise). Diligently follow all
waste disposal regulations.
2.1 Plant Material
1. Field or greenhouse-grown plants of appropriate age and life
stage, preferably with fully expanded leaves, should be used (see
Notes 1 and 2). The protocol is optimized for grapevine (Vitis
vinifera) and coffee (Coffea arabica) leaves. Approximately
25 g of fresh weight was used for grapevine (around 30 fully
expanded leaves), and about 10 g of fresh weight was used for
coffee (around 8 pairs of leaves).
2. Paper towel.
3. Scalpel.
50
Andreia Figueiredo and Leonor Guerra-Guimara ˜ es
protocol is a crucial step in a proteomic approach, and subsequent
compatibility with the analytical techniques (e.g., mass spectrometry) has to be considered.
The most commonly used technique for plant APF extraction is
the vacuum infiltration-centrifugation (VIC), described by Klement in 1965 [6]. The VIC involves two critical steps: vacuum
infiltration with appropriate extraction buffer and centrifugation
[7]. The composition of the infiltration buffer is one of the key
features of this method, as it should combine the maintenance of
osmotic pressure to prevent plasma membrane damage and cytoplasm content leaking, and the efficiency to extract cell wall proteins
[7]. High concentrations of ionic reagents, such as KCl, NaCl, or
LiCl, are normally considered to guarantee weakly bound cell wall
protein solubilization [2]. The centrifugation step is also limiting,
as centrifugation speed may cause damage to cell walls and
membranes [7].
Here, we present a vacuum protocol optimized for woody plant
leaves, namely Vitis vinifera and Coffea arabica. This protocol is
relatively easy to perform, allows obtaining an acceptable protein
yield, avoids contamination with cytosolic components, and is
compatible with high-throughput mass spectrometry-based proteomic approaches, namely shotgun proteomics. We discuss critical
points within the extraction procedure, namely the influence of the
composition of the infiltration buffer. Using this protocol, we have
isolated apoplastic proteins from both coffee and grapevine leaves
[8–10], yielding a good coverage of the apoplast proteome.
2 Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water, to attain a sensitivity of 18 MΩ cm at 25
C) and
analytical-grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise). Diligently follow all
waste disposal regulations.
2.1 Plant Material
1. Field or greenhouse-grown plants of appropriate age and life
stage, preferably with fully expanded leaves, should be used (see
Notes 1 and 2). The protocol is optimized for grapevine (Vitis
vinifera) and coffee (Coffea arabica) leaves. Approximately
25 g of fresh weight was used for grapevine (around 30 fully
expanded leaves), and about 10 g of fresh weight was used for
coffee (around 8 pairs of leaves).
2. Paper towel.
3. Scalpel.
50
Andreia Figueiredo and Leonor Guerra-Guimara ˜ es