Chapter 8 Making a Protein Extract from Plant Pathogenic Fungi for Gel- and LC-Based Proteomics Raquel González Fernández, Inmaculada Redondo, and Jesus V. Jorrin-Novo Abstract Proteomic technologies have become a successful tool to provide relevant information on fungal biology. In the case of plant pathogenic fungi, this approach would allow a deeper knowledge of the interaction and the biological cycle of the pathogen, as well as the identification of pathogenicity and virulence factors. These two elements open up new possibilities for crop disease diagnosis and environment-friendly crop protection. Phytopathogenic fungi, due to its particular cellular characteristics, can be considered as a recalcitrant biological material, which makes it difficult to obtain quality protein samples for proteomic analysis. This chapter focuses on protein extraction for gel- and LC-based proteomics with specific protocols of our current research with Botrytis cinerea. Key words Fungal proteomics, Fungal secretome, Gel-based proteomics, LC-based proteomics

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Introduction Phytopathogenic fungi are one of the most damaging plant parasitic organisms that cause serious diseases and remarkable yield losses in crops. The biological study of these microorganisms and the interaction with their hosts have experienced great advances in recent years due to the development of modern, holistic, and high-throughput -omic techniques, together with the increasing number of genome sequencing projects and the development of mutants and reverse genetics tools. Within these -omic techniques, proteomics has become a relevant tool in plant–fungus pathosystem research (reviewed in [1–4]). Molecular studies of the fungal biological cycle and their interaction with their hosts are necessary for searching key protein targets, and for developing new more efficient and environment-friendly agrochemicals [5, 6], which may open new ways for crop disease diagnosis and protection. Thus, proteomics aims to identify gene products with a key role in pathogenicity and virulence.

Jesus V. Jorrin-Novo et al. (eds.), Plant Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 1072, DOI 10.1007/978-1-62703-631-3_8, © Springer Science+Business Media, LLC 2014

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The use of proteomics also allows location-specific analyses (i.e., subproteomes at the level of organelles, cell membranes, cell wall, secretory proteins), the study of posttranslational modifications [7], as well as the study of interactions of host–pathogen [8] or host– pathogen-biocontrol agents [9, 10]. Proteomics involves the combined application of (a) advanced gel-based separation, such as one- and two-dimensional electrophoresis (1-DE and 2-DE), or gel-free based in liquid chromatography (LC) techniques; (b) identification techniques based on mass-spectrometry (MS) analysis; and (c) bioinformatics tools to characterize the proteins in complex biological mixtures [3, 4]. Different fields can be defined in proteomics, including descriptive and differential expression proteomics. In the case of fungi, a new area can also be defined as secretomics (the secretome is defined as the combination of native proteins and cell machinery involved in their secretion), since many fungi secrete an arsenal of proteins to accommodate their saprotrophic lifestyle, such as proteins implicated in the adhesion to the plant surface, host–tissue penetration, and invasion effectors, together with other virulence factors [11]. Over the last years, there has been a great advance in fungal proteomic research due to the availability of powerful proteomics technologies and the increasing number of fungal genome sequencing projects. Currently, more than 20 plant pathogenic fungal genomes have been sequenced (Broad Institute Database, http://broadinstitute.org/science/project/fungalgenomeinitiative), and excellent reviews of fungal proteomics methodologies have been recently published [3, 4, 12]. The workflow of a fungal gel-based proteomics experiment includes, among others, the following steps (Fig. 1): experimental design, fungal growth, sampling, sample preparation, protein extraction, separation, MS analysis, protein identification, statistical analysis of data, quantification, and data analysis, management, and storage. Most plant pathogenic fungi are filamentous and can be considered, just like plants, as recalcitrant biological material due to their robust cell wall accounting for 50–60 % of polysaccharides (glucans), 20–30 % of glycoproteins (mannoproteins), and 10–20 % of chitin [13]. Protein sample preparation is a critical step. The cell breakdown and later protein extraction are difficult because of the presence of a cell wall that makes up the majority of the cell mass [13]. Cell disruption can be performed using mechanical lysis via glass beads [14–16], with a cell mill [17] or by sonication [18–20]. These methods are more efficient than those based on chemical or enzyme extraction [21]. An alternative approach to avoid the difficulty of lysing the fungal cell wall might be the generation of protoplasts (cells whose wall has been completely or partially removed using either mechanical or enzymatic means) [22]. The most widely used method for cell disruption is pulverizing the mycelium in liquid nitrogen using a mortar and pestle [3, 4].

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Fig. 1 Overview of proteomics workflow in plant pathogenic fungi

The production of high-quality protein samples is also crucial for proteomic analysis. The most widely employed protocol for fungal proteins uses protein precipitation media containing organic solvents, such as trichloroacetic acid (TCA), followed by solubilization of the

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precipitate in an appropriate buffer. This method minimizes protein degradation/modification. Furthermore, it removes interfering compounds such as polysaccharides, polyphenols, pigment, and lipids, which may cause problems during IEF [23], and prevents protease activities [24]. TCA treatment complicates subsequent protein solubilization for IEF, especially with hydrophobic proteins. These problems have been partially overcome by the use of chaotropes (urea and thiourea) [25], new zwitterionic detergents [26–30], and a brief treatment with sodium hydroxide [24] that led to an increase in resolution and capacity of 2-DE gels. Other protein extraction methods have reported an improvement when using an acidic extraction solution to reduce streaking of fungal samples caused by their cell wall [31], as well as with the use of a phosphate buffer solubilization before the precipitation [32, 33]. 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 [34–36]. Alternative protocols for protein extraction from spores of Aspergillus ssp. have been optimized, since they use acidic conditions, step organic gradient, and variable sonication treatments (ultrasonic homogenizer and sonic water bath) [20]. Special protocols are required for secreted proteins due to the fact that there may be problems like a very low protein concentration that are sometimes below the detection limit of colorimetric methods (Bradford, Lowry, or BCA), or the presence of polysaccharides, mucilaginous material, salts, and secreted metabolites (lowmolecular organic acids, fatty acids, phenols, quinones, 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 [37]. This determination can also be affected by the high concentration of reagents from the solubilization buffer (i.e., 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 vary in the order of two magnitudes [38]. Comparisons of different standard methods for protein precipitation have demonstrated their limited applicability in analyzing the whole fungal secretome [39–44]. Electrophoresis is the most employed technique for protein separation in fungal research. Despite its simplicity, 1-DE remains as a useful technique that provides relevant information, especially in the case of comparative proteomics with large number of samples to analyze. It is possible by using this technique to distinguish between phenotypes of different wild-type strains of B. cinerea, and to identify proteins involved in the pathogenicity mechanisms (Fig. 2) [45, 46]. With appropriate software tools, 1-DE is a simple, reliable technique for fingerprinting of crude extracts, and it is especially useful in the case of hydrophobic and low-molecularweight proteins [47].

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Fig. 2 Protein profiles of seven B. cinerea wild-type strains (B05.10, T4, 2100, 2850, 2996, 20518, and BOCL) that differ both in host and virulence from (a) mycelium and (b) secreted proteins. This approach allows the assessment of differences in protein band patterns among strains

Two-DE is the dominant platform in fungal proteomics. The 2-DE consists of a tandem 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, the proteins are separated according to their approximate molecular weight using SDS-PAGE. Excellent reviews describing and discussing the features and protocols of electrophoretic separations in proteomics strategies have been published [23, 48]. Two main advantages of 2-DE can be emphasized: (a) its high protein separation capacity, and (b) the possibility of making largescale protein-profiling experiments. Nevertheless, the 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 [49]. After separating proteins, they can be detected using different staining techniques [23, 48], namely, (a) organic dyes, like colloidal Coomassie Blue staining, (b) zinc-imidazole staining, (c) silver staining, and (d) fluorescence-based detection, like Sypro Ruby. The criteria used to choose the staining method are the level of sensibility and its compatibility with MS. Gels are digitized, and bands or spots are studied by specific image analysis software (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 chromatography 2-D LC-MS/MS (based on a high-performance chromatofocusing in the first dimension followed by highresolution reversed-phase chromatography in the second) [50], and 1-DE-nanoscale capillary LC-MS/MS, like GeLC-MS/MS

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(this technique combines a size-based protein separation with an in-gel digestion of the resulting fractions) [51]. This GeLC-MS/ MS strategy paves the way towards the analysis on large-scale fungal response environmental cues on the basis of quantitative shotgun protein-profiling experiments. The case of multidimensional protein identification technology (MudPIT), which allows the identification of a much larger number of proteins compared to gel-based methods, has been a drawback due to the lack of quantitative data [52, 53]. MudPIT was used to analyze germling growth mechanisms in Uromyces appendiculatus by comparing germinating asexual uredospores to inactive spores [54]. MS is the basic technique for global proteomic analysis due to its accuracy, resolution, and sensitivity (in the femtomole to attomole concentration range), and due to the fact that it has the capacity for a high throughput. Not only does it allow profiling a proteome, but also and more important, it allows the identification of protein species and the characterization of posttranslational 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 proteomics). Protein species are identified by comparison of the experimental spectra, while the theoretical ones were obtained in silico from protein, genomic, EST sequence, or MS spectra databases. For this purpose, different instrumentation, algorithms, databases, and repositories are available [55, 56]. Although 2-DE remains as a standard tool for fungal proteomic research, current efforts are focussed on alternative gel-free shotgun strategies to identify and quantify proteins. The coupling of nanoscale separations (nanocapillary or nLC) with automated MS/MS has enhanced the development of this methodology. Using LC MS/MS, the complex mixtures of proteins are digested to peptides (normally with trypsin), which are separated according to their hydrophobicity by nLC, and then the eluted peptides are introduced into the mass spectrometer [57]. For example, a gelfree analysis from mycelium and secreted proteins of B. cinerea B05.10 and T4 strains has been carried out using a SYNAPT HDMS mass spectrometer (Waters) interfaced with a NanoAcquity UPLC System (Waters) [45, 59, 60]. A total of 197 and 73 proteins were identified from mycelia and secreted proteins, respectively (Fig. 3). Recent reviews study techniques, software, and statistical analyses used in gel-free quantitative proteomics, and discuss about its strengths and limitations [3, 61, 62]. Although several methods for proteomic analysis of limited fungal species have been published [4, 12, 63, 64], procedures for protein extraction as well as gel-based and gel-free analysis conditions are progressively evolving to study individual characteristics of fungal species.

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Fig. 3 Venn diagram of B05.10 versus T4 B. cinerea strains of proteins identified by using gel-free approach from (a) mycelium extracts and (b) secreted proteins

2 2.1

Materials Fungal Strains

2.2 Reagents, Solutions, and Buffers

This protocol has been carried out with different B. cinerea strains: B05.10 and T4 (provided by Dr. Julia Schumacher, Prof. Dr. Paul Tudzynski of the Institute of Biology and Biotechnology of Plants, Westfälische Wilhelms-Universität, Münster, Germany), together with CECT2100, CECT2850, CECT2996, and CECT20518 (provided by the Spanish Type Culture Collection). Analytical grade reagents are used, unless other grades are specified. The prepared solutions are kept at 4 °C or −20 °C if indicated. Reagents and solutions must be discarded once used, according to current regulations. It is mandatory to use a fume cupboard when working with volatile or dangerous compounds. Personal protection elements (i.e., lab coats, gloves, glasses) must be used (see Note 1). 1. 10 % (w/v) TCA in 80 % (v/v) acetone. 2. 0.1 M ammonium acetate in 100 or 80 % methanol. 3. 80 % (v/v) acetone. 4. Phenol solution equilibrated with 10 mM Tris–HCl, pH 8 (P4557, Sigma). 5. SDS buffer: 0.1 M Tris–HCl, pH 8, 30 % (w/v) sucrose, 2 % (w/v) SDS, 5 % (v/v) β-mercaptoethanol. 6. Solubilization solution: 9 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 0.5 % (v/v) Triton X-100, 20 mM DTT. 7. Bradford solution (B6916, Sigma). 8. Extraction buffer: 50 mM Tris–HCl, pH 8, 8 M urea, 1 % (w/v) SDS, 1 mM EDTA, 100 mM DTT. 9. TE buffer for secreted proteins: 10 mM Tris–HCl, pH 8, 1 mM EDTA, 2 % (v/v) β-mercaptoethanol, 1 mM PMSF, 10 μL/mL buffer of protease inhibitor cocktail for fungi (P8215, Sigma).

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10. Electrophoresis buffer: 50 mM Tris–HCl, pH 8, 192 mM glycine, 1 % (w/v) SDS. 11. Stain-Free Precast Gels (Criterion System, BioRad): 4–20 % Tris– HCl multi-wells for 1-DE and 8–16 % Tris–HCl IPG+1 for 2-DE. 12. IPG strips, 11 cm, pH 5–8 (BioRad). 13. IPG strip rehydration solution: 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 2 % (v/v) ampholytes (BioRad), 20 mM DTT. 14. Equilibration buffer: 1.5 M Tris–HCl, pH 8.8, 6 M urea, 20 % (v/v) glycerol, 2 % (w/v) SDS. 15. Distilled water. 16. Liquid nitrogen. 2.3

Equipment

1. Freeze-dryer. 2. Mortar and pestle. 3. Cell strainer, 100 μm nylon. 4. Vortexer. 5. Micropestles. 6. Ultrasonic homogenizer. 7. Microcentrifuge and centrifuge. 8. Disposable microcentrifuge tubes: 1.5 and 2.0 mL. 9. Centrifuge tubes: 50 mL. 10. Microtube mixer. 11. Criterion™ Cell (Biorad). 12. Criterion Stain Free Imager and Image Lab™ software (Biorad). 13. Shaker. 14. GS-800™ Calibrated Densitometer and Quantity One® 1-D Analysis software (BioRad).

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Methods The methods described below have been optimized to mycelium, secreted proteins in liquid media, and conidia from Botrytis cinerea, although these procedures can be applied to proteomic analysis of filamentous fungi in general.

3.1 Sample Collection

For in vitro cultures, conidia are produced using rich-media plates at 22 °C under constant black light (UV) during 3–4 weeks. Mycelium and secreted proteins can be obtained from liquid cultures inoculated with conidia or non-sporulating mycelia (see Note 2). Mycelia and media can be separated by centrifugation and filtration, frozen in liquid nitrogen, or lyophilized. At least three biological replicates should be collected [65].

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3.2 Protein Extraction by TCA/ Acetone–Phenol/ Methanol

Protein extraction is carried out by using the TCA/acetone–phenol/ methanol method [36, 66] with some modifications [12, 45], and adapted to the initial material (conidia, mycelium, or secreted proteins).

3.2.1 Mycelium

The lyophilized mycelium is ground to a fine powder in liquid nitrogen using a cooled mortar and pestle, and then it is stored at −80 °C for later analysis (see Note 3). The following protocol is used for protein extraction: 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 firstly using a micropestle, and secondly 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 the pellet at room temperature to remove residual acetone. 11. Add 1.2 mL of 1:1 phenol (pH 8, SIGMA)/SDS buffer. Mix well by vortexing and using a pipette. Incubate for 5 min on ice. 12. Centrifuge at 16,000 × g for 5 min. Transfer the upper phenol phase 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 incubate 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.

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19. Air-dry the pellet at room temperature. 20. Dissolve the proteins with a solubilization solution by shaking for 2 h in a microtube mixer at 4 °C (see Note 6). 21. Quantify proteins using the Bradford method [67]. 22. Store the protein extracts at −20 °C for further analysis. 3.2.2 Secreted Proteins

Lyophilized media are re-solubilized in 5 mL of TE buffer, and proteins are precipitated using the following protocol: 1. Transfer the re-solubilized medium into a 50-mL tube and add 2/1 (v/v) (10 mL) of 20 % (w/v) TCA/acetone. Mix well by vortexing and allow protein precipitation overnight 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 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 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. 7. Air-dry the pellet at room temperature to remove residual acetone. 8. Add 1.5 mL of 1/1 (v/v) phenol (pH 8, Sigma)/SDS buffer. Mix well by vortexing and transfer the results into a new 1.5mL Eppendorf tube. Incubate for 5 min on ice. 9. Centrifuge at 16,000 × g for 10 min. Transfer the upper phenol phase into a new 2-mL tube. 10. Fill the tube with 0.1 M ammonium acetate in 100 % (v/v) methanol, mix well, and allow the precipitation overnight at −20 °C. 11. Follow the steps in Subheading 3.2.1 (starting from step 15).

3.2.3 Conidia

Conidia can be harvested from H2O with 0.01 % Tween-80 scraping on the surface of an agar plate. The conidial suspension is filtered through a cell strainer, concentrated in 1.5-mL tubes, centrifuged at 16,000 × g for 5 min (4 °C), lyophilized, and stored at −80 °C for further analyses. For protein extraction, a bufferbased extraction is used and the proteins are precipitated using the TCA/acetone–phenol/methanol method [36, 66] with some modifications [12, 20]: 1. Add 300 μL of buffer extraction to conidia. Mix well firstly using a micropestle, and secondly by vortexing. 2. Sonicate 3 × 10 s (50 W, amplitude 60), breaking on ice at 1 min. Mix well firstly using a micropestle, and secondly by vortexing.

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3. Centrifuge at 16,000 × g for 5 min (4 °C). 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 discard the supernatant. 6. Fill the tube with 0.1 M ammonium acetate in 80 % (v/v) methanol. Mix well using firstly a micropestle, and secondly by vortexing. 7. Follow the steps in Subheading 3.2.1 (starting from step 5). 3.3 Protein Samples for LC

The fungal protein extracts obtained by the TCA/acetone–phenol/ methanol method are re-solubilized in a solubilization solution that contains thiourea, CHAPS, and Triton X-100. These compounds are important for protein solubilization, but they interfere with many downstream analysis methods (as is the case of LC separation). For this reason, it is crucial to remove them. Several methods to solve this problem are described below: 1. Protein solubilization with a solution of 6 M urea and 50 mM (NH4)2CO3 instead of the solubilization solution (see Note 8). 2. Use of cleaning kits (e.g., 2-D Clean-up kit; GE Healthcare), and protein re-solubilization in detergent-free solutions (i.e., 6 M urea, 50 mM (NH4)2CO3) (see Note 8). 3. Use of electrophoresis using 5 % acrylamide gels. Proteins (about 50–100 μg) are loaded onto the gel, run at 100 V, stained using the Coomassie method, and finally the protein band is cut out (see Note 9).

3.4 Protein Separation by 1-DE

Proteins can be separated by SDS-PAGE with the Laemmli electrophoresis system [68], for example using the Criterion System (BioRad) with precast Criterion Stain-Free precast Gels, Tris–HCl, and 4–20 % linear gradient (Bio-Rad). The 1-DE is visualized using the Image Lab System (Bio-Rad), and stained by Coomassie Blue Brilliant (CBB) method [69] (see Note 10). After protein staining, bands can be analyzed using the Quantity One software (Bio-Rad).

3.5 Protein Separation by 2-DE

Focusing conditions will vary with sample composition, complexity, and strip pH range. In our conditions, the 11 cm IPG strips, pH 5–8 (Bio-Rad), are rehydrated with 50 μg of protein extract in rehydration solution according to the manufacturer’s instructions, and applying 50 V for 16 h (active rehydration) at 20 °C. Before this focusing, a wet wick is placed under each end of the strip (cathode). The conditions for IEF have been adapted to our system from [38]: 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 20,000 V·h at 8,000 V, with a cell temperature of 20 °C (see Note 11). After IEF, IPG strips are stored at −20 °C.

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Before the second dimension, IPG strips are equilibrated following two steps. Firstly, it is carried out with 2 % (w/v) DTT in equilibration buffer for 10 min in agitation at room temperature; secondly, 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 explained in Subheading 3.3, but using Criterion Stain-Free precast Gels, Tris–HCl, and 8–16 % linear gradient IPG strips (Bio-Rad). After protein staining, spots can be analyzed using the PD-Quest software (Bio-Rad). 3.6 Protein Separation by LC

Protein sample (obtained according to Subheading 3.2) is digested at 37 °C overnight using trypsin. Tryptic peptides are cleaned and concentrated in a C18 Cartridges Octadecyl C18/18 % (Applied Separations), dried in a Speed-vac® Concentrator, and re-suspended in 50 μL of a 5 % ACN and 0.1 % formic acid solution. Finally, peptides are injected to the LC system (i.e., a Finnigan Surveyor an HPLC system). All these processes were made according to the Proteomic Service protocols of SCAI, University of Córdoba.

3.7 Protein Identification

The bands or spots are cut out and digested with trypsin. Tryptic peptides are analyzed in a mass-spectrometer (i.e., a 4800 Proteomics Analyzer MALDI–TOF/TOF, Applied Biosystems). In this case, the eight most abundant peptide ions are chosen for an MS/MS analysis. A PMF search and a combined search (+MS/ MS) are performed using the nrNCBI database of proteins with the MASCOT algorithm (www.matrixscience.com). All these processes were made according to Proteomic Service protocols of SCAI, University of Córdoba (see Note 12).

3.7.1 Gel-Based Techniques

3.7.2 Gel-Free Techniques

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Peptides are detected for example in an LTQ-Orbitrap equipped with a nanoelectrospray ion source (nESI). The acquired data can be analyzed with Proteome Discoverer v1.3 software (Thermo Fisher Scientific, USA) and MASCOT (http://www.matrixscience.com) or SEQUEST (http://fields.scripps.edu/sequest/) algorithms, using the public Botrytis cinerea Database from Botrytis cinerea Sequencing Project of Broad Institute of Harvard and MIT (http://www.broadinstitute.org/annotation/genome/botrytis_ cinerea/MultiHome.html), according to Proteomic Service protocols of SCAI, University of Córdoba (see Note 13).

Final Remarks With the use of gel-based techniques, around 50 bands and 500 spots were resolved subjected to 1- and 2-DE, respectively, from B. cinerea B05.10 mycelia protein extracts [45]. With the use of label-free LC-based approach, around 240 protein species were

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identified. A total of 224 of these identified proteins were also quantified, taking into account only those with at least 3 detected peptides [59]. Summarizing, we have shown in this chapter several protocols to work with different fungal material based on our experience with the phytopathogenic fungus B. cinerea. Moreover, the use of complementary proteomic approaches, as gel-based (1-DE and 2-DE) and gel-free (label-free LC-based) techniques, provides higher proteome coverage, relevant information being obtained on fungal biology and their interaction with their hosts [3, 4, 12, 45, 59].

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Notes 1. Gloves should be used for these procedures, and special care should be taken when handling TCA and phenol (consult safety data sheets) since they are corrosive products. Steps involving phenol and β-mercaptoethanol should be performed in a fume hood. 2. Examples of rich-media are PDAB (potato, dextrose, agar + bean leaves), solid synthetic complete medium (CM) [70], or solid malt extract medium (1.5 % w/v). 3. Be careful when working with liquid nitrogen: due to its cool temperature (−195.8 °C) it may cause severe frostbite. The nitrogen evaporates reducing the concentration of oxygen in the air and may act as an asphyxiating agent, especially in confined spaces. Remember that it may be dangerous because nitrogen is odorless, colorless, and tasteless, and it could cause suffocation without any sensation or warning. 4. Be careful: do not throw out the pellet. 5. Three phases appear, namely, the upper phase (which is the phenolic phase where proteins are), a white interphase, and a lower aqueous phase. Try not to get parts from the white interphase. 6. The volume of solubilization solution added will depend on the quantity of precipitated proteins. It is advisable that samples be well concentrated. 7. In this case, the precipitated pellet may be faint because the proteins secreted to the medium are at a very low concentration. 8. The problem derived from the use of solutions without thiourea, CHAPS, and Triton X-100 is the loss of hydrophobic proteins. 9. Acrylamide gel (5 %) (for 10 mL monomer solution): Mix 5.7 mL of ddH2O, 1.7 mL of 30 % (v/v) acrylamide–bisacrylamide BioRad mixture, 2.5 mL of 0.5 M Tris–HCl pH 6.8 buffer,

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and 100 μL of 10 % (w/v) SDS. Immediately prior to pouring the gel into the hand cast for Mini-PROTEAN two-gel electrophoresis system (BioRad), add 50 μL of 10 % (w/v) APS and 5 μL of TEMED and mix gently. The use of electrophoresis to remove it would lead to a reduced loss of these proteins. 10. More details about 1-DE and 2-DE separation methods are described in two excellent reviews [23, 48]. 11. The condition of protein focusing must be optimized for each study method. In our case, we use the PROTEAN IEF cell by Bio-Rad. The conditioning phase involves the application of previous steps at low voltage that allow to remove ions and other contaminants containing the sample, and that interfere on protein focusing. The current should not exceed 50 μA per strips. For more information see the 2-D Electrophoresis for Proteomics Manual by Bio-Rad. 12. More details about the MS analysis are described in [56, 64, 71–73]. 13. The Botrytis cinerea Database was downloaded from the Botrytis cinerea Sequencing Project, Broad Institute of Harvard and MIT (http://www.broadinstitute.org/annotaMore tion/genome/botrytis_cinerea/MultiHome.html). details about gel-free analysis are described in [59, 74, 75].

Acknowledgements This work was supported by the Spanish Ministry of Science and Innovation (BotBank Project, EUI2008-03686), the Andalusian Regional Government (Junta de Andalucía), and the University of Córdoba (AGR-0164: Agricultural and Plant Biochemistry and Proteomics Research Group). References 1. Afroz A, Ali GM, Mir A et al (2011) Application of proteomics to investigate stress-induced proteins for improvement in crop protection. Plant Cell Rep 30:745–763 2. Quirino BF, Candido ES, Campos PF et al (2010) Proteomic approaches to study plant-pathogen interactions. Phytochemistry 71:351–362 3. Gonzalez-Fernandez R, Jorrin-Novo JV (2012) Contribution of proteomics to the study of plant pathogenic fungi. J Proteome Res 11:3–16 4. Gonzalez-Fernandez R, Prats P, Jorrin-Novo JV (2010) Proteomics of plant pathogenic fungi. J

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Making a protein extract from plant pathogenic fungi for gel- and LC-based proteomics.

Proteomic technologies have become a successful tool to provide relevant information on fungal biology. In the case of plant pathogenic fungi, this ap...
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