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Laboratory Protocols in Fungal Biology

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 Proteomic Protocols for the Study
of Filamentous Fungi 24
Raquel González Fernández
and Jesús V. Jorrín Novo

Abstract
In the last few years, proteomics has experienced rapid improvement in
technologies and applications. Gel-based strategies have become the
method of choice for both identification and quantification of proteins in
most studies. The workflow of a standard gel-based proteomic experiment
includes experimental design, sampling, protein extraction, protein sepa-
ration, mass spectrometry analysis, protein identification, data statistical
analysis, validation of the identification, quantification, and data analysis.
The appropriate protocol to be used depends on and must be optimized for
the biological system (i.e., fungal species, plant species, organ, tissue,
cells). Preliminary steps are relevant. The choice of a good extraction pro-
tocol in a proteomic experiment is crucial because only if you can extract
and solubilize a protein you have a chance of detecting and identifying it.
This is more important in the case of filamentous fungi, which, owing to
their particular cellular characteristics, can be considered recalcitrant bio-
logical material, making it difficult to obtain quality protein samples to
proteomic analysis.
Fungi have an exceptionally robust cell wall, consisting largely of chi-
tin, which makes up the majority of the cell mass. Because of its rigidity,
cell lysis is an important element in fungal proteomics. For protein extrac-
tion, various buffer- and precipitation-based protocols are available. In
most of these protocols, trichloroacetic acid (TCA) and/or acetone are
used for protein precipitation, or a phenol extraction is made, where pro-
teins are solubilized in the phenolic phase and then are precipitated with
methanol and ammonium sulfate. These methods also eliminate some

R.G. Fernández (*) • J.V.J. Novo

Department of Biochemistry and Molecular Biology,
University of Córdoba, Agro-forestry and Plant
Biochemistry and Proteomics Research Group,
Ed. Severo Ochoa, planta baja. Campus de Rabanales,
Córdoba 14071, Spain
e-mail: q42gofer@uco.es

V.K. Gupta et al. (eds.), Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology, 299
Fungal Biology, DOI 10.1007/978-1-4614-2356-0_24, © Springer Science+Business Media, LLC 2013
300 R.G. Fernández and J.V.J. Novo

contaminants abundant in fungal material (such as polysaccharides, lipids,

nucleic acids, or phenolic compounds) that affect the protein isoelectrofo-
cusing and electrophoresis processes.

Key words
fungal proteomics • fungal secretome • cell lysis • protein precipitation
• protein isoelectrofocusing

also be defined as secretomics (the secretome is

Introduction
In a post-genomic era, proteomic technologies
have become a powerful tool to study the pro-
teome and to examine alterations in protein
profiles [1]. Similar to genomics and transcrip-
tomics, proteomics has evolved to incorporate
high-throughput processes, which allow faster
analysis of a larger number of proteins [2, 3].
Proteomics involves the combined applications
of advanced separation gel-based, namely mono-
and two-dimensional electrophoresis (1-DE and
2-DE), and gel-free, such as liquid chromatogra-
phy (LC) techniques, identification techniques
such as mass spectrometry (MS) analysis and
bioinformatics tools to characterize the proteins
in complex biological mixtures [4].
Plant pathogenic fungi cause significant yield
losses in crops. Molecular studies of the fungal
biological cycle and their interaction with their
hosts are necessary, in order to develop efficient
and environment-friendly crop protection strate-
gies [5, 6]. Proteomics, in combination with other
techniques, constitutes a successful tool for pro-
viding important information about pathogenic-
ity and virulence factors. Moreover, proteomics
also allows location-specific analysis (i.e., sub-
proteomes at the level of organelles, cell mem-
branes, cell wall, secretory proteins, etc.), the
study of post-translational modifications [7] and
interactions of host-pathogen, as well as host-
pathogen-biocontrol agents [8, 9]. As a conse-
quence, proteomics is opening up new possibilities
for crop disease diagnosis and crop protection.
Several areas can be defined in proteomics,
including descriptive and differential expression
proteomics. In the case of fungi, a new area can
defined as being the combination of native pro-
teins and cell machinery involved in their secre-
tion), since many fungi secrete an arsenal of
proteins to accommodate their saprotrophic life-
style, namely proteins implicated in the adhesion
to the plant surface, host-tissue penetration, and
invasion effectors, together with other virulence
factors [10]. Fungal proteomics research has
experienced great advances over the last years,
because of the availability of powerful proteom-
ics technologies and the increasing number of
fungal genome sequencing projects. Currently,
more than 50 pathogenic fungal genomes have
been sequenced.1 Excellent reviews on fungal
proteomic methodologies have been recently
published [4, 11]. The workflow of a fungal gel-
based proteomics experiment includes, among
others, the following steps: experimental design,
fungal growth, sampling, sample preparation,
protein extraction, separation, MS analysis, pro-
tein identification, statistical analysis of data,
quantification, and data analysis, management,
and storage.
Most of plant pathogenic fungi are filamentous
fungi. This type of fungi can be considered, simi-
larly to plants, recalcitrant biological material, so
the preparation of protein samples is a critical
step. Cell breakdown and protein extraction are
difficult because of the presence of a cell wall
that makes up the majority of the cell mass [12].
To overcome this challenge, early studies were
performed using mechanical lysis via glass beads
[13–15], a cell mill [16], or sonication [17–19],
because these methods are more efficient than
1
Broad Institute Database, http://broadinstitute.org/
science/project/fungal-genomeinitiative
24 Proteomic Protocols for the Study of Filamentous Fungi
those based on chemical or enzyme extraction
[20]. Shimizu and Wariishi [21] employed an
alternative approach to avoid the difficulty of
lysing the fungal cell wall by generating proto-
plasts of Tyromyces palustris. A better 2-DE pat-
tern was obtained from protoplast than from
intact cells. The most widely used method for
cell disruption consists of pulverizing the myce-
lium in liquid nitrogen using a mortar and pestle
[17, 22–33]. The production of high-quality pro-
tein samples is also a crucial step for proteomic
analysis. The protocol most widely employed for
fungal proteins uses protein precipitation media
containing organic solvents, such as trichloroa-
cetic acid (TCA), followed by solubilization of
the precipitate in an appropriate buffer. This
method minimizes protein degradation/
modification. Furthermore, it removes interfering
compounds such as polysaccharides, polyphe-
nols, pigment and lipids, which may be a prob-
lem during IEF [34], and prevents protease
activities [35]. TCA-treatment complicates sub-
sequent protein solubilization for IEF, especially
with hydrophobic proteins. These problems have
been partly overcome by the use of chaotropes
(urea and thiourea) [36], new zwitterionic deter-
gents [37–41], and by a brief treatment with
sodium hydroxide [35], which led to an increase
in resolution and capacity of 2-DE gels. Other
protein extraction methods have reported an
improvement when using acidic extraction solu-
tion to reduce streaking of fungal samples caused
by their cell wall [42], as well as using a phos-
phate buffer solubilization before the precipita-
tion [23, 24]. Finally, the combined use of TCA
precipitation and phenol extraction provides a
better spot definition, due to the fact that it reduces
streaking and leads to a higher number of detected
spots [22, 43]. Alternative protocols for protein
extraction from spores of Aspergillus ssp. have
been optimized, since they use acidic conditions,
step organic gradient, and variable sonication
treatment (ultrasonic homogenizer and sonic
water bath) [19].
Special protocols are required for secreted pro-
teins, due to the fact that there may be problems
such as a very low protein concentration, some-
times below the detection limit of colorimetric
301
methods (Bradford, Lowry, or BCA), or the
presence of polysaccharides, mucilaginous
material, salts, and secreted metabolites (low-
molecular organic acids, fatty acids, phenols, qui-
nones, and other aromatic compounds). The
presence of these extracellular compounds may
impair standard methods for protein quantification
and may result in a strong overestimation of total
protein number [44]. This determination can also
be affected by the high concentration of reagents
from the solubilization buffer (such as urea,
thiourea, or DTT) that may interfere in the
spectrophotometric measurement, producing an
overestimation of the total amount of protein in
which, depending on the method, the differences
varied in the order of two magnitudes [45].
Comparison of different standard methods for
protein precipitation has demonstrated their lim-
ited applicability to analyzing the whole fungal
secretome [45–54].
Electrophoresis is almost the only protein sep-
aration technique employed in fungal research.
Despite its simplicity, 1-DE remains as quite a
valid technique providing relevant information,
especially in the case of comparative proteomics
with large numbers of samples to analyze. Thus,
it is possible by using this technique to distin-
guish between phenotypes of different wild-type
strains of Botrytis cinerea and to identify proteins
involved in the pathogenicity mechanisms
(Gonzalez-Fernandez et al., personal communi-
cation). With appropriate software, 1-DE is a
simple, reliable technique for finger-printing
crude extracts, and it is especially useful in the
case of hydrophobic and low-molecular-weight
proteins [53]. Therefore, the 1-DE is a good
approach to obtain preliminary results before car-
rying on 2-DE analysis [54–58].
Two-DE is the dominant platform in fungal
proteomics. Briefly, the 2-DE consists of a tan-
dem pair of electrophoretic separations: in the
first dimension, proteins are resolved according
to their isoelectric points (pIs), normally using
IEF; while in the second dimension, proteins are
separated according to their approximate molec-
ular weight using SDS-PAGE. Excellent reviews
describing and discussing the features and proto-
cols of electrophoretic separations in proteomics
302
strategies have been published [34, 59]. The main
advantages of 2-DE are its high protein separa-
tion capacity and the possibility of making large-
scale protein-profiling experiments. Nevertheless,
reproducibility and resolution of this technique
are still remaining challenges. This method was
reported to under-represent proteins with extreme
physicochemical properties (size, isoelectric
point, transmembrane domains), as well as those
with a low abundance [60].
After separating proteins, they can then be
detected by a variety of staining techniques [34,
59] namely: (1) organic dyes, such as colloidal
Coomassie blue staining; (2) zinc–imidazole
staining; (3) silver staining; and (4) fluorescence-
based detection, such as Sypro Ruby. The crite-
ria to choose the staining method are the level of
sensibility and its compatibility with MS. Gels
are digitalized, and bands or spots are studied by
specific software of image analysis (i.e.,
Quantity-One, PD-Quest, BioRad). Bands or
spots are excised from gels and prepared for MS
analysis.
The limitations of gel-based analysis have led
to the more recent development of techniques
based on LC separation of proteins or peptides,
including two-dimensional liquid-phase chroma-
tography 2-D LC-MS/MS (based on a high per-
formance chromatofocusing in the first dimension
followed by high-resolution reversed-phase chro-
matography in the second) [61], and one-dimen-
sional electrophoresis(1-DE)-nanoscale capillary
LC-MS/MS, namely GeLC-MS/MS (this tech-
nique combines a size-based protein separation
with an in-gel digestion of the resulting fractions)
[62]. This GeLC-MS/MS strategy paves the way
toward the analysis on a large-scale fungal
response environmental cues on the basis of
quantitative shotgun protein- profiling experi-
ments. The case of Multidimensional Protein
Identification Technology (MudPIT), which
allows the identification of a much larger number
of proteins compared to gel-based methods, is
drawback being the lack of quantitative data [2,
63]. MudPIT was used to analyze the mecha-
nisms of germling growth in Uromyces appen-
diculatus by comparing germinating asexual
uredospores with inactive spores [64].
R.G. Fernández and J.V.J. Novo
MS is the basic technique for global proteomic
analysis due to its accuracy, resolution, and sen-
sitivity (in the femtomole to attomole concentra-
tion range), and due to the fact that is has the
capacity for a high throughput. Not only does it
allow one to profile a proteome, but more impor-
tantly, it allows one to identify the protein spe-
cies and characterize post-translational
modifications and interactions. Proteins are
identified from mass spectra of intact proteins
(top-down proteomics), or peptide fragments
obtained after enzymatic (mostly digested with
trypsin) or chemical treatment (bottom-up pro-
teomics). Protein species are identified by com-
parison of the experimental spectra, while the
theoretical ones were obtained in silico from pro-
tein, genomic, ESTs sequence, or MS spectra
databases. For that purpose, different instrumen-
tation, algorithms, databases, and repositories
are available [65, 66].
Although methods for proteomic analysis of
limited fungal species have been published [4,
11, 67–69], procedures for protein extraction and
2-DE gel analysis conditions are progressively
evolving according to individual characteristics
of fungal species.
Materials
(See Note 1)
1. Distilled water.
2. Liquid nitrogen.
3. Freeze-dryer.
4. Mortar and pestle.
5. Cell strainer, 100 mm Nylon (BD Falcon).
6. Vortexer.
7. Micropestles.
8. Ultrasonic homogenizer.
9. Microcentrifuge and centrifuge.
10. Disposable microcentrifuge tubes: 1.5 mL
and 2 mL.
11. Centrifuge tubes: 50 mL.
12. Trichloroacetic acid (TCA) (10% w/v)/ace-
tone (80% v/v) solution.
13. 0.1 M Ammonium acetate/methanol (100%
and 80% v/v) solution.
14. Acetone (80% v/v) solution.
24 Proteomic Protocols for the Study of Filamentous Fungi
15. Phenol solution equilibrated with 10 mM
Tris–HCl pH 8 (Sigma-Aldrich).
16. SDS buffer: 30% (w/v) sucrose, 2% (w/v)
SDS, 5% (v/v) b-mercaptoethanol, 0.1 M
Tris–HCl pH 8.
17. Solubilization solution: 9 M urea, 2 M thio-
urea, 4% (w/v) CHAPS, 0.5% (v/v)
Tritón-X100, 20 mM DTT.
18. Microtube mixer.
19. Bradford solution (Sigma-Aldrich).
20. Extraction buffer: 8 M urea, 1% (w/v) SDS,
1 mM EDTA, 100 mM DTT, 50 mM Tris–HCl
pH 8.
21. TE buffer for secreted proteins: 50 mM EDTA,
2% (v/v) b-mercaptoethanol, 1 mM PMSF,
10 mL/mL buffer of protease inhibitor cocktail
for fungi (Sigma-Aldrich), Tris–HCl pH 8.
22. Running buffer: 192 mM Glycine, 1% (w/v)
SDS, 50 mM Tris–HCl pH 8.
23. Vertical electrophoresis equipment; for
example, Criterion System (BioRad).
24. Precast free stain gels (Criterion System,
BioRad): 4–20% Tris–HCl multi-wells for
1-DE and 8–16% Tris–HCl IPG + 1 for 2-DE.
25. IPG strips, 11 cm, pH 5–8 (BioRad).
26. IPG strips rehydration solution: 7 M urea,
2 M thiourea, 4% (v/v) CHAPS, 2% (v/v)
ampholytes (BioRad), 20 mM DTT.
27. Equilibration buffer: 6 M urea, 20% (v/v)
glycerol, 2% (w/v) SDS, 1.5 M Tris–HCl pH
8.8.
28. Shaker.
29. Densitometer; for example, GS-800
(BioRad).
Methods
The methods below have been optimized to
mycelium, secreted proteins in liquid media, and
conidia from B. cinerea, although these proce-
dures can be applied in proteomic analysis of
filamentous fungi in general.
Sample Collection
For in vitro cultures, conidia are produced using
rich-media plates at 22 °C under constant black
303
light (UV) for 3–4 weeks. Mycelium and secreted
proteins can be obtained from liquid cultures
inoculated with conidia or nonsporulating myce-
lia (see Note 2). Mycelia and media can be sepa-
rated by centrifugation and filtration, frozen in
liquid nitrogen, and lyophilized.
Protein Extraction by TCA/
Acetone-Phenol/Methanol Method
Protein extraction is carried on by using the TCA/
acetone–phenol/methanol method [70, 71] with
some modifications [4] and adapted to started
material (conidia, mycelium or secreted
proteins).
Mycelium
The lyophilized mycelium is ground to a fine
powder in liquid nitrogen using a cooled mortar
and pestle, and stored at −80 °C for later analysis
(see Note 3). For protein extraction, the follow-
ing protocol is applied:
1. Transfer 50–100 mg of mycelial powder into
a 2-mL tube.
2. Add 1 mL of 10% (w/v) TCA/acetone and
mix well using a micropestle and then by
vortexing.
3. Sonicate 3 × 10 s (50 W, amplitude 60) at
4 °C, breaking on ice at 1 min.
4. Fill the tube with 10% (w/v) TCA/acetone.
Mix well by vortexing.
5. Centrifuge at 16,000 × g for 5 min (4 °C) and
remove the supernatant by decanting (see
Note 4).
6. Fill the tube with 0.1 M ammonium acetate
in 80% (v/v) methanol. Mix well by
vortexing.
7. Centrifuge at 16,000 × g for 5 min (4 °C) and
discard the supernatant.
8. Fill the tube with 80% (v/v) acetone. Mix
well by vortexing.
9. Centrifuge at 16,000 × g for 5 min (4 °C) and
discard the supernatant.
10. Air-dry at room temperature to remove resid-
ual acetone.
11. Add 1.2 mL of 1:1 phenol (pH 8, SIGMA)/
SDS buffer. Mix well using a pipette and by
vortexing. Incubate for 5 min in ice.
304
12. Centrifuge at 16,000 × g for 5 min. Transfer
the upper phenol fase into a new 1.5-mL tube
(see Note 5).
13. Fill the tube with 0.1 M ammonium acetate
in 100% (v/v) methanol, mix well, and allow
the precipitation overnight at −20 °C.
14. Centrifuge at 16,000 × g for 5 min (4 °C) and
discard the supernatant (a white pellet should
be visible).
15. Wash the pellet with 100% methanol and
mix by vortexing.
16. Centrifuge at 16,000 × g for 5 min (4 °C) and
discard the supernatant.
17. Wash the pellet with 80% (v/v) acetone and
mix by vortexing.
18. Centrifuge at 16,000 × g for 5 min (4 °C) and
discard the supernatant.
19. Dry the pellet at room temperature.
20. Dissolve the proteins in solubilization solu-
tion for 2 h, shaking in a microtube mixer at
4 °C (see Note 6).
21. Quantify proteins using Bradford method
[72].
22. Store the protein extracts at −20 °C for
further analysis.
Secreted Proteins
Lyophilized media are re-suspended in 5 mL of
TE buffer and proteins are precipitated according
to the following protocol:
1. Transfer the medium resolubilized into a
50-mL tube and add 2/1 (v/v) (10 mL) of
20% (w/v) TCA/acetone. Mix well by vor-
texing and allow protein precipitation over-
night at 4 °C.
2. Centrifuge at 16,000 × g for 10 min (4 °C)
and remove the supernatant by decanting
(see Note 7).
3. Add a volume 4/1 (v/v) (20 mL) of 0.1 M
ammonium acetate in 80% (v/v) methanol.
Mix well by vortexing.
4. Centrifuge at 16,000 × g for 10 min (4 °C)
and discard the supernatant.
5. Add a volume 4/1 (v/v) (20 mL) of 80% (v/v)
acetone. Mix well by vortexing.
6. Centrifuge at 16,000 × g for 10 min (4 °C)
and discard the supernatant.
R.G. Fernández and J.V.J. Novo
7. Air-dry at room temperature to remove
residual acetone.
8. Add 4 mL of 1/1 (v/v) phenol (pH 8, SIGMA)/
SDS buffer. Mix well by vortexing and trans-
fer the 4-mL to two 2-mL eppendorf. Incubate
for 5 min in ice.
9. Centrifuge at 16,000 × g for 10 min. Transfer
the upper phenol phase into a new 2-mL tube
(1 mL per 2-mL tube).
10. Fill the tube with 0.1 M ammonium acetate
in 100% (v/v) methanol, mix well, and allow
to precipitate overnight al −20 °C.
11. Centrifuge one 2-mL tube at 16,000 × g
for 5 min (4 °C) and discard the supernatant
(a slight pellet should be visible). Fill the
same 2-mL tube with the other eppendorf
(mix well before changing). Centrifuge at
16,000 × g for 5 min (4 °C) and discard the
supernatant.
12. Follow the steps in Mycelium section, start-
ing with step 15.
Conidia
Conidia can be harvested with H2Od with 0,01%
Tween-80 scraping on the surface of agar plate.
The conidia suspension is filtered through a cell
strainer, concentred in 1.5-mL tubes, centri-
fuged at 16,000 × g for 5 min (4 °C), lyophilized
and stored at −80 °C for further analysis. For
protein extraction, the TCA/acetone-phenol/
methanol [70, 71] method was used, with some
modifications [4, 19].
1. Add 300 mL of extraction buffer to conidia. Mix
well using a micropestle and by vortexing.
2. Sonicate 3 × 10 s (50 W, amplitude 60), break-
ing on ice at 1 min. Mix well using a micrope-
stle and by vortexing.
3. Centrifuge at 16,000 × g for 5 min (4 °C).
4. Fill the tube with 10% (w/v) TCA/acetone.
Mix well using by vortexing.
5. Centrifuge at 16,000 × g for 5 min (4 °C) and
discard the supernatant.
6. Fill the tube with 0.1 M ammonium acetate in
80% (v/v) methanol. Mix well using a
micropestle and by vortexing.
7. Follow the steps in Mycelium, starting with
step 5.
24 Proteomic Protocols for the Study of Filamentous Fungi
Protein Separation
One-Dimensional Electrophoresis
Proteins can be separated by SDS-PAGE accord-
ing to Laemmly electrophoresis system, [73] for
example, using Criterion System (BioRad) with
precast Criterion Stain Free Gels, Tris–HCl,
4–20% linear gradient (BioRad). The 1-DE is
visualized using the Image Lab System (BioRad),
and stained by CBB (Coomassie Blue Brilliant)
method [74] (see Note 8). After the staining of
proteins, bands can be analyzed using the
Quantity-One software (BioRad).
Two-Dimensional Electrophoresis
Isoelectrofocusing
Focusing conditions will vary with sample compo-
sition, sample complexity, and strip pH range. In
our conditions, the 11 cm IPG strips, pH 5–8
(BioRad), are rehydrated with 50 mg of protein
extract in 185 mL rehydration solution applying
50 V for 16 h (active rehydratation) at 20 °C. Before
the focusing a wet wick is inserted under each end
of the strip (catode). The conditions for IEF have
been adapted to our system from reference [45]:
150 V for 1 h, 1 h at 200 V, 1 h at 500 V, 1,000 V·h
at 1,000 V, followed by 2.5 h gradient from 1,000 to
8,000 V, and finally focused for 30,000 V·h at
8,000 V, with a cell temperature of 20 °C (see
Note 9). After IEF, IPG strips are stored at −20 °C.
Before the second dimension, IPG strips are
equilibrated in two steps. Firstly, it is carried on
with 2% (w/v) DTT in equilibration buffer for
10 min in agitation at room temperature; sec-
ondly, it is done with 2.5% (w/v) iodoacetamide
in equilibration buffer for 10 min in agitation at
room temperature.
The second dimension is performed in the
same way as SDS-PAGE, but using precast
Criterion Stain Free Gels, Tris–HCl, 8–16% lin-
ear gradient for IPG strips (BioRad). After the
staining of proteins, spots can be analyzed using
the PD-Quest software (BioRad).
Protein Identification
The bands or spots are cut out and digested with
trypsin. Tryptic peptides are analyzed in a mass
305
spectrometer; for example, a 4,800 Proteomics
Analyzer MALDI–TOF/TOF (Applied
Biosystems). In this case, the most abundant pep-
tide ions are subjected to MS/MS analysis.
A PMF search and a combined search (+MS/MS)
are performed in nrNCBI database of proteins
using the MASCOT algorithm (see Note 10).
Notes
1. Gloves and lab coat should be used in these
procedures, and particular care should be taken
when handling TCA and phenol (consult safety
data sheets) because they are corrosive prod-
ucts. Steps involving phenol and b-mercapto-
ethanol should be performed in a fume hood.
2. Examples of rich-media are PDAB (potato,
dextrose, agar + bean leaves), solid synthetic
complete medium (CM) [75], or solid malt
extract medium (1.5% w/v).
3. Be careful to work with liquid nitrogen
because its cool temperature (−195.8 °C)
could cause severe frostbite. The nitrogen
evaporated reduces the concentration of oxy-
gen in the air and can act as an asphyxiant,
especially in confined spaces, so it may be
dangerous because nitrogen is odorless, col-
orless, and tasteless, and could cause suffo-
cation without any sensation or warning.
4. Be careful to not throw out the pellet.
5. Three phases appear, namely: the upper
phase (which is the phenolic phase where are
the proteins), a white interphase, and a lower
aqueous phase. Try to not to get part of the
white interphase.
6. The volume of solubilization solution added
will depend on quantity of precipitated pro-
teins. It is advisable that samples are well
concentrated.
7. In this case, maybe that the precipitated pel-
let is faint because the proteins secreted to
medium are at very low concentration.
8. More details about 1-DE and 2-DE separa-
tion methods are described in two excellent
reviews [34, 59].
9. The condition of protein focusing must be
optimized for each system of study. In our
case, we use the PROTEAN IEF cell by
306
BioRad. The conditionating phase involves
the application of previous steps at low volt-
age that allow to remove ions and other con-
taminants containing the sample, and that
interfere on protein focusing. The current
should not exceed 50 mA per strips. For more
information see the 2-D Electrophoresis for
Proteomics Manual by BioRad.
10. More details about MS analysis are described
in references [65, 68, 76].
Acknowledgments This work was supported by the
Spanish Ministry of Science and Innovation (BotBank
Project, EUI2008-03686), the Regional Government of
Andalusia (Junta de Andalucía) and the University of
Córdoba (AGR-0164: Agricultural and Plant Biochemistry
and Proteomics Research Group).
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