Chapter 49 Proteotyping of Holm Oak (Quercus ilex subsp. ballota) Provenances Through Proteomic Analysis of Acorn Flour José Valero Galván, Raquel González Fernández, Luis Valledor, Rafael Ma. Navarro Cerrillo, and Jesus V. Jorrin-Novo Abstract Proteomics has become a powerful tool to characterize biodiversity and natural variability in plant species, as well as to catalogue and establish phylogenetic relationships and distances among populations, provenances or ecotypes. In this chapter, we describe the standard proteomics workflow that we currently use in cataloguing Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) populations. Proteins are extracted from acorn flour or pollen by TCA/acetone or TCA/acetone-phenol methods, resolved by one- or twodimensional gel electrophoresis, and gel images are captured and analyzed by appropriate software and statistical packages. Quantitative or qualitative variable bands or spots are subjected to MS analysis in order to identify them and correlate differences in the protein profile with the phenotypes or environmental conditions. Key words Holm oak proteomics, Plant biodiversity, Plant proteotyping
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Introduction Electrophoresis has proven to be the most important and effective tool in analyzing cellular protein profiles both from a quantitative and qualitative point of view. Moreover, it is one of the most convenient methods for characterizing, cataloguing, and establishing phylogenetic relationships and distances among populations, provenances, ecotypes, or genotypes [1–8]. The workflow of a standard 1- or 2-DE-based proteomics experiment includes the following steps: experimental design, sampling, protein extraction, protein separation, protein analysis by mass spectrometry (MS), statistical analysis of the data, and protein identification, using proper bioinformatics software and tools [9–11] (Fig. 1). The experimental design is the key step to extracting the maximum information from an experiment. A good experimental design considers the impact of different sources of variation and the minimum number of replicates to be made in the context of a particular minimum detectable
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_49, © Springer Science+Business Media, LLC 2014
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Fig. 1 Overview of the Holm oak proteotyping workflow
difference that one is seeking to achieve [12]. The most appropriate protocols for protein extraction should be developed for each specific species, and they must be optimized for the biological systems (i.e., plant species, organ, tissue, cells), as well as for the
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research objectives [13]. In our experience, protein extraction using the TCA/acetone and TCA/acetone–phenol protocols provided the best results with a large variety of plant species [13–17]. Protein solubilization is a critical step. Detergents as CHAPS and chaotropic agents such as urea and thiourea must be used in the solubilization solutions to help in the hydrophobic protein solubilization, obtaining a higher protein yield [9, 16–19]. Protein separation with SDS-PAGE is a quick and accurate technique for plant proteins, especially in the case of comparative proteomics with large numbers of samples to be analyzed. Using appropriate software, SDS-PAGE 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 [20]. Furthermore, SDS-PAGE is a good approach to obtain preliminary results before performing 2-DE analysis [15, 16]. Two-DE has been used for separating and displaying the components of large protein complexes, and it has been a reliable tool to study natural variability in several plants species [1, 3–5, 21, 22]. After staining (we currently use colloidal Coomassie staining [23]), images are digitized and analyzed with appropriate software [24]. Quantitative proteomics data are classically assessed by univariate statistics (t-test, Mann–Whitney, ANOVA, Kruskal–Wallis), but these methods increase the possibility of false positives, are negatively affected by the raw structure of proteomics data, and they cannot detect trends and protein relations [25–28]. On the other hand, the analysis employing multivariate approaches (i.e., Principal Components, Self Organizing Maps) are described to be more effective, because of its capacity to reduce the complexity of the data, predict trends and also for being less affected by data structure [26–28]. Furthermore, data analysis can be used to discriminate and establish phylogenetic relationships among populations and genotyping, as well as to correlate the profile with edaphoclimatic characteristics and morphometric parameters. Finally, major differential bands or spots are excised from gels and subjected, after tryptic digestion, to MS analysis and their identification [29, 30].
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Materials Mention of specific companies or pieces of equipment is not mandatory, and it does not represent an endorsement by the authors. All the chemicals should be of analytical grade.
2.1 Reagents, Solutions and Buffers 2.1.1 Protein Extraction
1. Liquid Nitrogen. 2. Trichloroacetic acid (TCA) (10 % w/v)/acetone (80 % v/v) solution. Store at −20 °C and use directly from the freezer. 3. 0.1 M Ammonium acetate/methanol (100 % and 80 % v/v) solution. Store at −20 °C and use directly from the freezer.
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4. Acetone (80 % v/v) solution. Store at −20 °C and use directly from the freezer. 5. Phenol solution equilibrated with 10 mM Tris–HCl, pH 8 (Sigma, Chemical Company). Store at 4 °C. 6. Sodium dodecyl sulfate (SDS) buffer: 0.1 M Tris–HCl, pH 8, 30 % (w/v) sucrose, 2 % (w/v) SDS, 5 % (v/v) β-mercaptoethanol. Store at 4 °C and temper prior to use. Add the β-mercaptoethanol just before use. 7. Solubilization solution: 9 M urea, 2 M thiourea, 4 % (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate (CHAPS), 0.5 % (v/v) Tritón-X100, 20 mM dithiothreitol (DTT). Store in 1 mL aliquots at −20 °C. Add the DTT just before use. 2.1.2 Protein Quantification
1. Bradford solution (Sigma, Chemical Company) (see Note 1). Store at 4 °C. 2. Protein standard: bovine serum albumin (BSA) at a concentration of 1 mg/mL in distilled water is used as a stock solution (see Note 2). Store at −20 °C.
2.1.3 One-Dimensional Gel Electrophoresis
1. 1.5 M Tris–HCl, pH 8.8. Store at 4 °C. 2. 0.5 M Tris–HCl, pH 6.8. Store at 4 °C. 3. 30 % Acrylamide/Bisacrylamide solution, 37.5:1 (Bio-Rad). Store at 4 °C. 4. Sodium dodecyl sulfate solution (SDS): 10 % (w/v) in water. Store at room temperature. 5. Ammonium persulfate solution (APS): 10 % (w/v) in water. Store at −20 °C (see Note 3). 6. N,N,N′,N′-Tetramethylethylenediamine (TEMED) (Sigma) (see Note 4). Store at room temperature. 7. Running buffer: 25 mM Tris–HCl, pH 8.3, 192 mM glycine, 0.1 % (w/v) SDS. Store at room temperature (see Note 5). 8. Laemmli buffer: 62.5 mM Tris–HCl, pH 6.8, 25 % (v/v) glycerol, 2 % (w/v) SDS, 0.01 % (w/v) bromophenol blue, 5 % (v/v) β-mercaptoethanol. Store at room temperature. Add the β-mercaptoethanol just before use. 9. Standard broad range molecular weight markers (Bio-Rad). Store at 4 °C.
2.1.4 Two-Dimensional Gel Electrophoresis
1. Immobilized pH gradient strips of pH range 5–10, and 17 cm length (Bio-Rad). 2. Rehydration solution: 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 0.5 % (v/v) anfolites, 20 mM DTT, and 0.01 % (w/v) bromophenol blue. Store in 1 mL aliquots at −20 °C. Add the DTT just before use.
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3. SDS-PAGE reagents are the same as described in Subheading 2.1.3. 4. Equilibration buffer I: 50 mM Tris–HCl, pH 8.8, 6 M urea, 20 % (v/v) glycerol, 2 % (w/v) SDS, and 2 % (w/v) DTT. Store at 4 °C and temper prior to use. Add the DTT before use. 5. Equilibration buffer II: 50 mM Tris–HCl, pH 8.8, 6 M urea, 20 % (v/v) glycerol, 2 % (w/v) SDS, and 135 mM iodoacetamide. Store at 4 °C and temper prior to use. Add iodoacetamide before use. 2.1.5 Colloidal Coomassie Blue G-250 Staining
1. Staining solution: Weigh 80 g ammonium sulfate, add 22.5 mL of 85 % phosphoric acid, and add 700 mL of water. Dissolve 1 g of Coomassie blue G-250 in 22 mL of water. Mix the two solutions, and then add 200 mL of methanol. Finally add water until 1,000 mL. Store at room temperature. 2. 0.1 M Tris–H3PO4. Store at 4 °C. 3. 25 % (v/v) methanol. Store at room temperature. 4. 20 % (p/v) ammonium sulfate. Store at room temperature.
2.1.6 Protein Identification
Protein identification was carried out according to the protocols of the Proteomics Service of SCAI at the University of Córdoba. 1. Differential spots. 2. Porcine trypsin (Promega). 3. Desalting cartridges Zip Tips C18 (Agilent Technologies). 4. Peptide calibration standard (Bruker Daltonics), consisting of a combination of peptides that provides a good calibration across a typical mass range between 1,000 and 3,500 Da. 5. 0.1 % (v/v) trifluoroacetic acid (TFA). 6. Ammonium bicarbonate. 7. Acetonitrile (ACN). 8. Trifluoroacetic acid (TFA). 9. α-cyano hydroxycinnamic acid. 10. MALDI mass spectrometry calibration standards. 11. Matrix solution: 4.7 mg/mL α-cyano-4-hydroxycinnamic acid (Sigma) in 70 % (v/v) ACN. 12. AnchorChip MALDI target (Bruker Daltonics).
2.2 Equipment and Software
1. Airtight polyethylene bags. 2. A knife. 3. Blade mill (Moulinex AD56 42). 4. Microsieve (Ø: 15 cm, 1 mm).
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5. Mortar and pestle. 6. Vortex. 7. Ultrasonic homogenizer. 8. Microcentrifuge. 9. Disposable microcentrifuge tubes: 1.5 and 2 mL. 10. PROTEAN II Cell and Protean Dodeca Cell (Bio-Rad, Hercules, USA). 11. Protean IEF Cell system (Bio-Rad, Hercules, USA). 12. Micro-tubes mixer. 13. Gel shaker. 14. PowerPac 300 Power Supply (Bio-Rad, Hercules). 15. GS-800™ Calibrated Imaging Densitometer (Bio-Rad). 16. Quantity One® 1-D Analysis software (Bio-Rad Hercules). 17. PD-Quest software v8.1 (Bio-Rad). 18. ProGest digestion station (Genomics Solution). 19. MALDI plates. 20. Automatic ProMs station (Genomic Solution). 21. Resin C18 microcolumn (ZipTip, Millipore). 22. 4800 Proteomics Analyzer (Applied Biosystems). 23. Autoflex mass spectrometer (Bruker Daltonics). 24. MASCOT search engine (Matrix Science Ltd., London; http://www.matrixscience.com).
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Methods
3.1 Plant Material Collection and Storage
3.1.1 Acorns
Plant material will be collected from different provenances or genotypes. It is important to do a large sampling in order to have the greatest diversity of populations or genotypes possible. Undamaged, homogeneous mature acorns or pollen are collected from at least ten different trees for each population. Once harvested, plant material is put in airtight polyethylene bag, and then stored at 4 ± 1 °C during no more than 12 h. 1. Immediately after arriving to the laboratory, and previously to protein extraction, a pool of 20 acorns per tree are scarified with a knife by making transversal and longitudinal cuts, permitting the rapid removal of the pericarp (see Note 6). 2. After being peeled out, their embryos (including cotyledons) are crushed in a blade mill (Moulinex AD56 42) until obtain a fine powder (flour).
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3. The final powder is weighted and stored in a desiccator at 4 ± 1 °C until protein extraction. 4. For SDS-PAGE protein extraction, flour of ten independent samples per population are used; whereas for 2-DE protein extraction flour representing all studied accessions of each population are crushed together and its proteins are extracted. 3.1.2 Pollen
1. Immediately after arriving to the laboratory, pollen grains are isolated from freshly open flowers by shaking the anthers on a glass slide (see Note 6). 2. Flower debris is removed using a microsieve, and pollen is examined under a light microscope. 3. Pollen is either used immediately, or stored at −70 °C after freezing in liquid nitrogen (see Note 7).
3.2 Protein Extraction from Acorns by the TCA/ Acetone Method
To extract protein from acorns, we propose to use the procedure suggested by Damerval et al. [14]. It is particularly suitable for the extraction of proteins from seeds, but also for leaves as reported by Jorge et al. [31] and Maldonado et al. [13]. The method described here has been optimized to leaves and seeds from Q. ilex subsp. ballota [15], although these procedures can be applied to plant proteomic analysis in general. 1. The powder (flour) (100 mg) is transferred into a 2 mL tube with 1 mL of a solution of 10 % (w/v) TCA/acetone with 0.07 % (w/v) DTT. Mix well using a micropestle and then by vortexing (see Notes 6 and 8). 2. Sonicate 3× 10 s (50 W, amplitude 60) at 4 °C. 3. The proteins are left to precipitate overnight and then centrifuged at 15,000 × g at 4 °C for 15 min. Discard the supernatant. 4. The pellet is washed twice with 1 mL of a solution of acetone with 0.07 % (w/v) DTT, and then centrifuged at 15,000 × g at 4 °C for 15 min. Discard the supernatant. 5. The pellet obtained is dried in the air in order to remove residual acetone (see Note 9). 6. Proteins are dissolved in a solubilization solution for 2 h, by shaking in a microtube mixer at 4 °C (see Note 10). 7. Proteins are quantified using the Bradford method [32]. Prepare the calibration curve using several dilutions of bovine serum albumin protein, containing concentration of 0, 1, 3, 5, 10, 15, and 20 μL of BSA (1 mg/mL) into 1.5 mL tubes, and make all up to 500 μL with distilled water. Add 500 μL of Bradford reagent to each tube and mix well by vortexing gently for thorough mixing. Use 800 μL of distilled water as control. Incubate the samples with the protein reagent at room
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temperature for 30 min in darkness. Measure the absorbance at 595 nm of each sample and standards. Store the protein extracts at −20 °C for further analysis. 3.3 Protein Extraction from Pollen by the TCA/Acetone– Phenol Method
The TCA/acetone–phenol protocol provided the best results in terms of spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility [13]. We propose to use it to extract proteins from leaves and pollen [16, 31], although these procedures can also be applied to plant proteomic analysis in general. 1. The pollen (100 mg) is transferred into a 2 mL tube with 1 mL of a solution of 10 % (w/v) TCA/acetone. Mix well using a micropestle and then by vortexing (see Notes 6 and 8). 2. Sonicate 3× 10 s (50 W, amplitude 60) at 4 °C. 3. Fill the tube with the solution of 10 % (w/v) TCA/acetone. Mix well by vortexing and centrifuge at 16,000 × g at 4 °C for 5 min. Remove the supernatant by decanting. 4. Fill the tube with 0.1 M ammonium acetate in 80 % (v/v) methanol. Mix well by vortexing and centrifuge at 16,000 × g at 4 °C for 5 min. Discard the supernatant. 5. Fill the tube with a solution of 80 % (v/v) acetone. Mix well by vortexing and centrifuge at 16,000 × g at 4 °C for 5 min. Discard the supernatant. 6. Air-dry the pellet at room temperature to remove residual acetone (see Note 9). 7. 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 on ice and centrifuge at 16,000 × g for 5 min. Transfer the upper phenol phase into a new 1.5-mL tube (see Note 11). 8. Fill the tube with a solution of 0.1 M ammonium acetate in 100 % (v/v) methanol, mix well and complete the precipitation overnight at −20 °C. 9. Centrifuge at 16,000 × g at 4 °C for 5 min and discard the supernatant (a white pellet should be visible). 10. Wash the pellet with 100 % (v/v) methanol and mix by vortexing. Centrifuge at 16,000 × g at 4 °C for 5 min and discard the supernatant. 11. Wash the pellet with 80 % (v/v) acetone and mix by vortexing. Centrifuge at 16,000 × g at 4 °C for 5 min and discard the supernatant. 12. Dry the pellet at room temperature. 13. Dissolve the proteins in the solubilization solution for 2 h, shaking in a microtube mixer at 4 °C (see Note 10). 14. Procedures for quantify proteins are the same as described in Subheading 3.2 (steps 7 and 8). Store the protein extracts at −20 °C for further analysis.
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Prepare the 13 % polyacrylamide gel electrophoresis using a PROTEAN II cells (Bio-Rad, Hercules, USA) electrophoresis kit. 1. Prepare the resolving gel solution by mixing 27.08 mL of 30 % of acrylamide, 15.6 mL of Tris–HCl, pH 8.8, 0.625 mL of SDS, 18.8 mL of distilled water, 0.31 μL of APS and 31.2 μL of TEMED. Pour the solution into the gel cassette and cover completely the solution surface with isopropanol to obtain a flat layer on top of the resolving gel. Leave at room temperature until 20 min. 2. When polymerization is completed, prepare the stacking gel. Mix 1.63 mL of 30 % acrylamide, 2.5 mL of Tris–HCl, pH 6.8, 0.1 mL of SDS, 6.75 mL distilled water, 50 μL APS and 10 μL TEMED and gently stir to obtain a uniform solution. Pour the resolving gel and transfer the well-forming comb into this solution. Polymerize the gel for at least 30 min at room temperature to allow complete polymerization. 3. The comb is removed from the stacking gel and place the gel in the electrophoresis tank. The gel is covered with running buffer, and 70 μL of sample is applied to the bottom of each well. The volume and protein concentration of the sample should be sufficient to give at least 50 μg of each protein. Apply 10 μL of the molecular weight standards to one or two wells, preferably in an asymmetric position. 4. Connect the wires to the power supply unit and apply 100 V until the blue dye front reaches the bottom of the gel. Disconnect the electrophoresis unit from the power supply, remove the lid and discard the running buffer. Remove the gel from plates with a spatula, discard the stacking gel, and wash the separated gel with distilled water to remove traces of running buffer. 5. Prepare the Coomassie brilliant blue G-250 staining 1 day before the staining process [23]. Place the gel in a tray containing 500 mL of staining solution. Incubate overnight the gel in the staining solution. Once the gel is stained, discard the staining solution and cover the gel with 0.1 M Tris–H3PO4. Then, shake for 1–3 min. Discard the solution and cover the gel with 25 % (v/v) of methanol. Then, shake for 1 min. Remove the methanol and wash the gel with 20 % (w/v) of ammonium sulfate for 24 h. 6. Images are digitized using a GS-800 Calibrated Densitometer (Bio-Rad, Hercules, USA) and analyzed with Quantity One software (Bio-Rad, Hercules, USA). 7. Excise the bands of interest from the gel, and place them in different tubes containing distilled water until their processing.
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3.5 Two-Dimensional Electrophoresis
Preliminary 2-DE experiments can be carried out with the MiniProtean 3 system (Bio-Rad, Hercules, USA), using 7 cm pH 3–10 linear gradient strips (Bio-Rad, Hercules, USA) and 13 % polyacrylamide gels, to examine the pI range where the proteins are concentrated. In our case, the most protein spots were located in a pI range between 5 and 8. 1. Prepare a mix with 300 μg of proteins in 250 mL of rehydration solution on 1.5 mL tube. Load the samples in each lane of the 17 cm strip holders. 2. Remove the protective cover from the surface of the IPG strips and slowly lower the IPG strip (gel slide down) onto the rehydration solution, without trapping air bubbles. Then cover the IPG strip with 1–2 mL of mineral oil and apply the plastic cover. 3. Apply a low voltage (50 V) during rehydration for 12 h at 20 °C for improving the entry of high molecular weight proteins [33]. 4. After active rehydration, start isoelectric focusing at 20 °C using the following parameters 250 V for 2 min, followed by 150 min linear gradient from 250 to 10,000 V, and finally focus on up to 40,000 V at 10,000 Vh. 5. After IEF, the strips are immediately reduced and alkylated according to [33]. IPG strips are equilibrated in two steps. Firstly, it is performed with 2 % (w/v) DTT in equilibration buffer I for 10 min in agitation at room temperature; secondly, it is carried out with 2.5 % (w/v) iodoacetamide in equilibration buffer II for 10 min in agitation at room temperature. 6. The second dimension is performed on 13 % polyacrylamide gels using the Protean Dodeca Cell (Bio-Rad, Hercules, USA). The gels can be run at 150 constant volts until the dye reaches the bottom of the gel. 7. The gels are stained employing the colloidal Coomassie method [23]. Soak the gel in a tray containing 50 mL of staining solution. Incubate overnight the gel in the staining solution. Once the gel is stained, discard the staining solution and cover the gel with 0.1 M Tris–H3PO4. Then, shake during 1–3 min. Discard the solution and cover the gel with 25 % (v/v) of methanol. Then shake during 1 min. Remove the methanol and wash the gel with 20 % (w/v) of ammonium sulfate for 24 h. 8. Images are digitized using a GS-800 Calibrated Densitometer (Bio-Rad, Hercules, USA), and then analyzed with PD-Quest software v8.1 (Bio-Rad, Hercules, USA).
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9. Select a good image as a reference with a clear and representative spot pattern and with a minimum distortion, and align each of the images to the chosen reference. Select between three prominent spots to manually assignment, and use the automatic vector tool to add additional vectors. 10. After automatic spot detection and matching, check manually the spots with edition tools for correct detection. 11. Set gel groups according to the experimental design and normalize spot volume intensity ratios for each spot. 12. List all the spots together with their normalized volume. 3.6 Phylogenetic and Statistical Analyses
Prior to statistical and phylogenetic analyses, the volume of pixels for each band or spot is normalized according to the total volume of bands detected (SDS-PAGE) or to the total volume of valid spots in each gel (2-DE), respectively. Then, they are logtransformed, following the recommendations described by Valledor and Jorrin [34]. A multivariate analysis is carried out on two steps: firstly, hierarchical clustering is performed to check the entire dataset, and the results are indicated in dendrograms using the cluster function of the software used; secondly, the dataset is analyzed by the use of Principal Component Analysis (PCA). The settings used for the PCA analysis are: co-variance matrix type, three principal components, onefold change, and 0.4 correlation threshold for clusters. PCA results are shown as a biplot. Since the employed statistical methods tend to classify population together, this information is necessary for studying the possible correlation between distances and geographical and climate parameters.
3.7 Protein Identification
Spots are manually excised with a scalpel. Protein identification was carried out according to the Proteomics Service protocols of the University of Córdoba. Gel plugs are digested with modified porcine trypsin (Sequencing grade; Promega), by using an automatic ProGest digestion station (Genomics Solution). The conditions are two detained steps for 30 min with 200 mM ammonium bicarbonate in 40 % (v/v) ACN at 37 °C; twice washed with 25 mM ammonium bicarbonate for 5 min and 25 mM ammonium bicarbonate in 50 % (v/v) ACN for 15 min respectively; dehydration with 100 % (v/v) ACN for 5 min and sample dried; hydration using 10 μL trypsin in a solution of 25 mM ammonium bicarbonate at a final concentration of 12.5 ng/μL for 10 min a room temperature, and the digestion is proceeded at 37 °C for 12 h. Subsequently, digestion is stopped by adding 10 μL of a solution of 0.5 % TFA in water.
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Tryptic peptides are purified in an automatic ProMS station (Genomic Solutions) by using a resin C18 microcolumn (ZipTip, Millipore), and they are eluted directly with a matrix solution (α-cyano hydroxycinnamic acid at a concentration of 5 mg/mL in 70 % (v/v) ACN/0.1 % (v/v) TFA) on MALDI plaque in 1 μL of final volume. After the cocrystallization on plaque, samples are analyzed by MALDI-TOF/TOF mass spectrometry to obtain the peptide mass fingerprinting (MS) in a 4800 Proteomics Analyzer (Applied Biosystems). The settings are: 800–4,000 m/z range, with an accelerating voltage of 20 kV, in reflection mode, with delayed extraction set to “on”, and an elapsed time of 120 ns. Spectra are internally calibrated with peptides from trypsin autolysis (M + H+ = 842.509, M + H+ = 2,211.104) with an m/z precision of ± 20 ppm. Most abundant peptide ions are subjected to MS/MS analysis, providing information that can be used to define the peptide sequence. A combined search (PMF and MS/MS) is performed with GPS ExplorerTM software v3.5 (Applied Biosystems) over nonredundant NCBI databases using the MASCOT search engine (Matrix Science Ltd., London; http://www.matrixscience.com). The database search utilized the following parameters: taxonomy restrictions to Viridiplantae, one missed cleavage sites, 100 ppm mass tolerance in MS and 0.5 Da for MS/MS data, cysteine carbamidomethylation as a fixed modification, and methionine oxidation as a variable modification. The confidence in the peptide mass fingerprinting matches (p < 0.05) is based on the MOWSE score, and confirmed by the accurate overlapping of the matched peptides with the major peaks of the mass spectrum.
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Final Remarks Taking into account our own experience, in this chapter we have shown the usefulness of a basic proteomic approach—based on SDS-PAGE and 2-DE analyses of protein extracts from mature seeds and pollen—for variability studies in Holm oak. These analyses have allowed the separation and grouping of the populations according to its acorn morphometry, location (northern and southern), and climate conditions (xeric, mesic, and intermediate), confirming previous results of our research group by using acorn morphometry and NIRS chemical composition [15, 16, 35]. Therefore, gel-based Proteomics (SDS-PAGE and 2-DE) can be used to detect variability between different populations from different environments, and to correlate it to environmental conditions (Fig. 1).
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Notes 1. Check the list of compatible chemicals and potential interfering chemicals typically found in the protein extraction buffer. 2. Do not freeze stock vial solutions more than once. 3. Prepare this solution on fresh each time. 4. TEMED accelerates the decomposition of APS molecules into sulfate free radicals and these, in turn, initiate the polymerization. 5. Prepare running buffer as 10× native buffer (0.25 M, Tris, 1.92 M glycine, and 1 % (w/v) SDS). Weigh 30.3 g of Tris, 144 g of glycine, and 10 g of SDS; mix and make it to 1 L with water. The pH of this solution should not be adjusted, and stored at room. 6. It is necessary to wear gloves and lab coat for all the procedures. 7. Be careful with liquid nitrogen due to its cool temperature (−195.8 °C). It could cause severe frostbite. Pay attention to laboratory safety regulation. 8. Be particularly careful when handling the reagents TCA and phenol (consult safety data sheets) because they are corrosive. Use the fume hood with volatile reagents. 9. Be careful in not throwing out the pellet. 10. The volume of the solubilization solution added will depend on the quantity of precipitated proteins. It is advisable that samples be well concentrated. 11. There are three phases, namely: the upper phase (which is the phenolic phase where the proteins are), a white interphase, and a lower aqueous phase. Try to not to take part of the white interphase.
Acknowledgments Jose Valero was recipient of an Alban Program fellowship (I06D00010MX). This work was supported by the Spanish Ministry of Science and Innovation cofinanced by the European Community FEDER funds: CGL2008-04503-C03-01/BOS, AGL2002-00530, and AGL2009-12243-C02-02; the Regional Government of Andalusia (Junta de Andalucía; the University of Córdoba (AGR-0164: Agricultural and Plant Biochemistry and Proteomics Research Group)); and the Autonomous University of Juarez City.
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different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis. J Proteomics 71:461–472 Damerval C, De Vienne D, Zivy M et al (1986) Technical improvements in twodimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis 7:52–54 Valero GJ, Valledor L, Navarro CRM et al (2011) Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) through acorn protein profile analysis. J Proteomics 74:1244–1255 Valero GJ, Valledor L, Fernández GR et al (2012) Proteomic analysis of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) pollen. J Proteomics 75:2736–2744 Wang W, Vignani R, Scali M et al (2006) A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. Electrophoresis 27:2782–2786 Jorrin JV, Maldonado AM, Castillejo MA (2007) Plant proteome analysis: a 2006 update. Proteomics 7:2947–2962 Jorrín-Novo JV, Maldonado AM, EchevarríaZomeño S et al (2009) Plant proteomics update (2007–2008): second-generation proteomic techniques, an appropriate experimental design, and data analysis to fulfill MIAPE standards, increase plant proteome coverage and expand biological knowledge. J Proteomics 72:285–314 Supek F, Peharec P, Krsnik-Rasol M et al (2008) Enhanced analytical power of SDSPAGE using machine learning algorithms. Proteomics 8:28–31 Petit RJ, Bahrman N, Baradat PH (1995) Comparison of genetic differentiation in maritime pine (Pinus pinaster Ait.) estimated using isozyme, total protein and terpenic loci. Heredity 75:382–389 Vienne DD, Burstin J, Gerber S et al (1996) Two-dimensional electrophoresis of proteins as a source of monogenic and codominant markers for population genetics and mapping the expressed genome. Heredity 76:166–177 Mathesius U, Keijzers G, Natera SH et al (2001) Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting. Proteomics 1:1424–1440
Holm Oak Proteotyping 24. Matthias B, Michael MF, Kolbe M et al (2007) The state of the art in the analysis of twodimensional gel electrophoresis images. Appl Microbiol Biotechnol 76:1223–1243 25. Chich JF, David O, Villers F (2007) Statistics for proteomics: experimental design and 2-DE differential analysis. J Chromatogr B 849:261–272 26. Rodríguez-Piñeiro AM, Rodríguez-Berrocal FJ, Páez de la Cadena M (2007) Improvements in the search for potential biomarkers by proteomics: application of principal component and discriminant analyses for two-dimensional maps evaluation. J Chromatogr 849:251–260 27. Grove H, Jørgensen BM, Jessen F (2008) Combination of statistical approaches for analysis of 2-DE data gives complementary results. J Proteome Res 7:5119–5124 28. Valledor L, Jorrín J (2011) Maximizing the information obtained by quantitative two dimensional gel electrophoresis analyses by an appropriate experimental design and statistical analyses. J Proteomics 74:1–18 29. Domon B, Aebersold R (2006) Mass spectrometry and protein analysis. Science 312:212–217 30. Han X, Aslanian A, Yates IJR (2008) Mass spectrometry for proteomics. Curr Opin Chem Biol 12:483–490
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31. Jorge I, Navarro RM, Lenz C et al (2005) The holm oak leaf proteome: analytical and biological variability in the protein expression level assessed by 2-DE and protein identification tandem mass spectrometry de novo sequencing and sequence similarity searching. Proteomics 5:222–234 32. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 33. Gorg A, Postel W, Baumer M et al (1992) Two-dimensional polyacrylamide gel electrophoresis, with immobilized pH gradients in the first dimension, of barley seed proteins: discrimination of cultivars with different malting grades. Electrophoresis 13:192–203 34. Valledor L, Jorrin J (2010) Back to the basics: maximizing the information obtained by quantitative two dimensional gel electrophoresis analyses by an appropriate experimental design and statistical analyses. J Proteomics 74:1–18 35. Valero GJ, Jorrín-Novo J, Gómez CA et al (2011) Population variability based on the morphometry and chemical composition of the acorn in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.). Eur J Forest Res 131:893–904
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Introduction Electrophoresis has proven to be the most important and effective tool in analyzing cellular protein profiles both from a quantitative and qualitative point of view. Moreover, it is one of the most convenient methods for characterizing, cataloguing, and establishing phylogenetic relationships and distances among populations, provenances, ecotypes, or genotypes [1–8]. The workflow of a standard 1- or 2-DE-based proteomics experiment includes the following steps: experimental design, sampling, protein extraction, protein separation, protein analysis by mass spectrometry (MS), statistical analysis of the data, and protein identification, using proper bioinformatics software and tools [9–11] (Fig. 1). The experimental design is the key step to extracting the maximum information from an experiment. A good experimental design considers the impact of different sources of variation and the minimum number of replicates to be made in the context of a particular minimum detectable
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_49, © Springer Science+Business Media, LLC 2014
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Fig. 1 Overview of the Holm oak proteotyping workflow
difference that one is seeking to achieve [12]. The most appropriate protocols for protein extraction should be developed for each specific species, and they must be optimized for the biological systems (i.e., plant species, organ, tissue, cells), as well as for the
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research objectives [13]. In our experience, protein extraction using the TCA/acetone and TCA/acetone–phenol protocols provided the best results with a large variety of plant species [13–17]. Protein solubilization is a critical step. Detergents as CHAPS and chaotropic agents such as urea and thiourea must be used in the solubilization solutions to help in the hydrophobic protein solubilization, obtaining a higher protein yield [9, 16–19]. Protein separation with SDS-PAGE is a quick and accurate technique for plant proteins, especially in the case of comparative proteomics with large numbers of samples to be analyzed. Using appropriate software, SDS-PAGE 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 [20]. Furthermore, SDS-PAGE is a good approach to obtain preliminary results before performing 2-DE analysis [15, 16]. Two-DE has been used for separating and displaying the components of large protein complexes, and it has been a reliable tool to study natural variability in several plants species [1, 3–5, 21, 22]. After staining (we currently use colloidal Coomassie staining [23]), images are digitized and analyzed with appropriate software [24]. Quantitative proteomics data are classically assessed by univariate statistics (t-test, Mann–Whitney, ANOVA, Kruskal–Wallis), but these methods increase the possibility of false positives, are negatively affected by the raw structure of proteomics data, and they cannot detect trends and protein relations [25–28]. On the other hand, the analysis employing multivariate approaches (i.e., Principal Components, Self Organizing Maps) are described to be more effective, because of its capacity to reduce the complexity of the data, predict trends and also for being less affected by data structure [26–28]. Furthermore, data analysis can be used to discriminate and establish phylogenetic relationships among populations and genotyping, as well as to correlate the profile with edaphoclimatic characteristics and morphometric parameters. Finally, major differential bands or spots are excised from gels and subjected, after tryptic digestion, to MS analysis and their identification [29, 30].
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Materials Mention of specific companies or pieces of equipment is not mandatory, and it does not represent an endorsement by the authors. All the chemicals should be of analytical grade.
2.1 Reagents, Solutions and Buffers 2.1.1 Protein Extraction
1. Liquid Nitrogen. 2. Trichloroacetic acid (TCA) (10 % w/v)/acetone (80 % v/v) solution. Store at −20 °C and use directly from the freezer. 3. 0.1 M Ammonium acetate/methanol (100 % and 80 % v/v) solution. Store at −20 °C and use directly from the freezer.
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4. Acetone (80 % v/v) solution. Store at −20 °C and use directly from the freezer. 5. Phenol solution equilibrated with 10 mM Tris–HCl, pH 8 (Sigma, Chemical Company). Store at 4 °C. 6. Sodium dodecyl sulfate (SDS) buffer: 0.1 M Tris–HCl, pH 8, 30 % (w/v) sucrose, 2 % (w/v) SDS, 5 % (v/v) β-mercaptoethanol. Store at 4 °C and temper prior to use. Add the β-mercaptoethanol just before use. 7. Solubilization solution: 9 M urea, 2 M thiourea, 4 % (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate (CHAPS), 0.5 % (v/v) Tritón-X100, 20 mM dithiothreitol (DTT). Store in 1 mL aliquots at −20 °C. Add the DTT just before use. 2.1.2 Protein Quantification
1. Bradford solution (Sigma, Chemical Company) (see Note 1). Store at 4 °C. 2. Protein standard: bovine serum albumin (BSA) at a concentration of 1 mg/mL in distilled water is used as a stock solution (see Note 2). Store at −20 °C.
2.1.3 One-Dimensional Gel Electrophoresis
1. 1.5 M Tris–HCl, pH 8.8. Store at 4 °C. 2. 0.5 M Tris–HCl, pH 6.8. Store at 4 °C. 3. 30 % Acrylamide/Bisacrylamide solution, 37.5:1 (Bio-Rad). Store at 4 °C. 4. Sodium dodecyl sulfate solution (SDS): 10 % (w/v) in water. Store at room temperature. 5. Ammonium persulfate solution (APS): 10 % (w/v) in water. Store at −20 °C (see Note 3). 6. N,N,N′,N′-Tetramethylethylenediamine (TEMED) (Sigma) (see Note 4). Store at room temperature. 7. Running buffer: 25 mM Tris–HCl, pH 8.3, 192 mM glycine, 0.1 % (w/v) SDS. Store at room temperature (see Note 5). 8. Laemmli buffer: 62.5 mM Tris–HCl, pH 6.8, 25 % (v/v) glycerol, 2 % (w/v) SDS, 0.01 % (w/v) bromophenol blue, 5 % (v/v) β-mercaptoethanol. Store at room temperature. Add the β-mercaptoethanol just before use. 9. Standard broad range molecular weight markers (Bio-Rad). Store at 4 °C.
2.1.4 Two-Dimensional Gel Electrophoresis
1. Immobilized pH gradient strips of pH range 5–10, and 17 cm length (Bio-Rad). 2. Rehydration solution: 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 0.5 % (v/v) anfolites, 20 mM DTT, and 0.01 % (w/v) bromophenol blue. Store in 1 mL aliquots at −20 °C. Add the DTT just before use.
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3. SDS-PAGE reagents are the same as described in Subheading 2.1.3. 4. Equilibration buffer I: 50 mM Tris–HCl, pH 8.8, 6 M urea, 20 % (v/v) glycerol, 2 % (w/v) SDS, and 2 % (w/v) DTT. Store at 4 °C and temper prior to use. Add the DTT before use. 5. Equilibration buffer II: 50 mM Tris–HCl, pH 8.8, 6 M urea, 20 % (v/v) glycerol, 2 % (w/v) SDS, and 135 mM iodoacetamide. Store at 4 °C and temper prior to use. Add iodoacetamide before use. 2.1.5 Colloidal Coomassie Blue G-250 Staining
1. Staining solution: Weigh 80 g ammonium sulfate, add 22.5 mL of 85 % phosphoric acid, and add 700 mL of water. Dissolve 1 g of Coomassie blue G-250 in 22 mL of water. Mix the two solutions, and then add 200 mL of methanol. Finally add water until 1,000 mL. Store at room temperature. 2. 0.1 M Tris–H3PO4. Store at 4 °C. 3. 25 % (v/v) methanol. Store at room temperature. 4. 20 % (p/v) ammonium sulfate. Store at room temperature.
2.1.6 Protein Identification
Protein identification was carried out according to the protocols of the Proteomics Service of SCAI at the University of Córdoba. 1. Differential spots. 2. Porcine trypsin (Promega). 3. Desalting cartridges Zip Tips C18 (Agilent Technologies). 4. Peptide calibration standard (Bruker Daltonics), consisting of a combination of peptides that provides a good calibration across a typical mass range between 1,000 and 3,500 Da. 5. 0.1 % (v/v) trifluoroacetic acid (TFA). 6. Ammonium bicarbonate. 7. Acetonitrile (ACN). 8. Trifluoroacetic acid (TFA). 9. α-cyano hydroxycinnamic acid. 10. MALDI mass spectrometry calibration standards. 11. Matrix solution: 4.7 mg/mL α-cyano-4-hydroxycinnamic acid (Sigma) in 70 % (v/v) ACN. 12. AnchorChip MALDI target (Bruker Daltonics).
2.2 Equipment and Software
1. Airtight polyethylene bags. 2. A knife. 3. Blade mill (Moulinex AD56 42). 4. Microsieve (Ø: 15 cm, 1 mm).
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5. Mortar and pestle. 6. Vortex. 7. Ultrasonic homogenizer. 8. Microcentrifuge. 9. Disposable microcentrifuge tubes: 1.5 and 2 mL. 10. PROTEAN II Cell and Protean Dodeca Cell (Bio-Rad, Hercules, USA). 11. Protean IEF Cell system (Bio-Rad, Hercules, USA). 12. Micro-tubes mixer. 13. Gel shaker. 14. PowerPac 300 Power Supply (Bio-Rad, Hercules). 15. GS-800™ Calibrated Imaging Densitometer (Bio-Rad). 16. Quantity One® 1-D Analysis software (Bio-Rad Hercules). 17. PD-Quest software v8.1 (Bio-Rad). 18. ProGest digestion station (Genomics Solution). 19. MALDI plates. 20. Automatic ProMs station (Genomic Solution). 21. Resin C18 microcolumn (ZipTip, Millipore). 22. 4800 Proteomics Analyzer (Applied Biosystems). 23. Autoflex mass spectrometer (Bruker Daltonics). 24. MASCOT search engine (Matrix Science Ltd., London; http://www.matrixscience.com).
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Methods
3.1 Plant Material Collection and Storage
3.1.1 Acorns
Plant material will be collected from different provenances or genotypes. It is important to do a large sampling in order to have the greatest diversity of populations or genotypes possible. Undamaged, homogeneous mature acorns or pollen are collected from at least ten different trees for each population. Once harvested, plant material is put in airtight polyethylene bag, and then stored at 4 ± 1 °C during no more than 12 h. 1. Immediately after arriving to the laboratory, and previously to protein extraction, a pool of 20 acorns per tree are scarified with a knife by making transversal and longitudinal cuts, permitting the rapid removal of the pericarp (see Note 6). 2. After being peeled out, their embryos (including cotyledons) are crushed in a blade mill (Moulinex AD56 42) until obtain a fine powder (flour).
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3. The final powder is weighted and stored in a desiccator at 4 ± 1 °C until protein extraction. 4. For SDS-PAGE protein extraction, flour of ten independent samples per population are used; whereas for 2-DE protein extraction flour representing all studied accessions of each population are crushed together and its proteins are extracted. 3.1.2 Pollen
1. Immediately after arriving to the laboratory, pollen grains are isolated from freshly open flowers by shaking the anthers on a glass slide (see Note 6). 2. Flower debris is removed using a microsieve, and pollen is examined under a light microscope. 3. Pollen is either used immediately, or stored at −70 °C after freezing in liquid nitrogen (see Note 7).
3.2 Protein Extraction from Acorns by the TCA/ Acetone Method
To extract protein from acorns, we propose to use the procedure suggested by Damerval et al. [14]. It is particularly suitable for the extraction of proteins from seeds, but also for leaves as reported by Jorge et al. [31] and Maldonado et al. [13]. The method described here has been optimized to leaves and seeds from Q. ilex subsp. ballota [15], although these procedures can be applied to plant proteomic analysis in general. 1. The powder (flour) (100 mg) is transferred into a 2 mL tube with 1 mL of a solution of 10 % (w/v) TCA/acetone with 0.07 % (w/v) DTT. Mix well using a micropestle and then by vortexing (see Notes 6 and 8). 2. Sonicate 3× 10 s (50 W, amplitude 60) at 4 °C. 3. The proteins are left to precipitate overnight and then centrifuged at 15,000 × g at 4 °C for 15 min. Discard the supernatant. 4. The pellet is washed twice with 1 mL of a solution of acetone with 0.07 % (w/v) DTT, and then centrifuged at 15,000 × g at 4 °C for 15 min. Discard the supernatant. 5. The pellet obtained is dried in the air in order to remove residual acetone (see Note 9). 6. Proteins are dissolved in a solubilization solution for 2 h, by shaking in a microtube mixer at 4 °C (see Note 10). 7. Proteins are quantified using the Bradford method [32]. Prepare the calibration curve using several dilutions of bovine serum albumin protein, containing concentration of 0, 1, 3, 5, 10, 15, and 20 μL of BSA (1 mg/mL) into 1.5 mL tubes, and make all up to 500 μL with distilled water. Add 500 μL of Bradford reagent to each tube and mix well by vortexing gently for thorough mixing. Use 800 μL of distilled water as control. Incubate the samples with the protein reagent at room
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temperature for 30 min in darkness. Measure the absorbance at 595 nm of each sample and standards. Store the protein extracts at −20 °C for further analysis. 3.3 Protein Extraction from Pollen by the TCA/Acetone– Phenol Method
The TCA/acetone–phenol protocol provided the best results in terms of spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility [13]. We propose to use it to extract proteins from leaves and pollen [16, 31], although these procedures can also be applied to plant proteomic analysis in general. 1. The pollen (100 mg) is transferred into a 2 mL tube with 1 mL of a solution of 10 % (w/v) TCA/acetone. Mix well using a micropestle and then by vortexing (see Notes 6 and 8). 2. Sonicate 3× 10 s (50 W, amplitude 60) at 4 °C. 3. Fill the tube with the solution of 10 % (w/v) TCA/acetone. Mix well by vortexing and centrifuge at 16,000 × g at 4 °C for 5 min. Remove the supernatant by decanting. 4. Fill the tube with 0.1 M ammonium acetate in 80 % (v/v) methanol. Mix well by vortexing and centrifuge at 16,000 × g at 4 °C for 5 min. Discard the supernatant. 5. Fill the tube with a solution of 80 % (v/v) acetone. Mix well by vortexing and centrifuge at 16,000 × g at 4 °C for 5 min. Discard the supernatant. 6. Air-dry the pellet at room temperature to remove residual acetone (see Note 9). 7. 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 on ice and centrifuge at 16,000 × g for 5 min. Transfer the upper phenol phase into a new 1.5-mL tube (see Note 11). 8. Fill the tube with a solution of 0.1 M ammonium acetate in 100 % (v/v) methanol, mix well and complete the precipitation overnight at −20 °C. 9. Centrifuge at 16,000 × g at 4 °C for 5 min and discard the supernatant (a white pellet should be visible). 10. Wash the pellet with 100 % (v/v) methanol and mix by vortexing. Centrifuge at 16,000 × g at 4 °C for 5 min and discard the supernatant. 11. Wash the pellet with 80 % (v/v) acetone and mix by vortexing. Centrifuge at 16,000 × g at 4 °C for 5 min and discard the supernatant. 12. Dry the pellet at room temperature. 13. Dissolve the proteins in the solubilization solution for 2 h, shaking in a microtube mixer at 4 °C (see Note 10). 14. Procedures for quantify proteins are the same as described in Subheading 3.2 (steps 7 and 8). Store the protein extracts at −20 °C for further analysis.
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Prepare the 13 % polyacrylamide gel electrophoresis using a PROTEAN II cells (Bio-Rad, Hercules, USA) electrophoresis kit. 1. Prepare the resolving gel solution by mixing 27.08 mL of 30 % of acrylamide, 15.6 mL of Tris–HCl, pH 8.8, 0.625 mL of SDS, 18.8 mL of distilled water, 0.31 μL of APS and 31.2 μL of TEMED. Pour the solution into the gel cassette and cover completely the solution surface with isopropanol to obtain a flat layer on top of the resolving gel. Leave at room temperature until 20 min. 2. When polymerization is completed, prepare the stacking gel. Mix 1.63 mL of 30 % acrylamide, 2.5 mL of Tris–HCl, pH 6.8, 0.1 mL of SDS, 6.75 mL distilled water, 50 μL APS and 10 μL TEMED and gently stir to obtain a uniform solution. Pour the resolving gel and transfer the well-forming comb into this solution. Polymerize the gel for at least 30 min at room temperature to allow complete polymerization. 3. The comb is removed from the stacking gel and place the gel in the electrophoresis tank. The gel is covered with running buffer, and 70 μL of sample is applied to the bottom of each well. The volume and protein concentration of the sample should be sufficient to give at least 50 μg of each protein. Apply 10 μL of the molecular weight standards to one or two wells, preferably in an asymmetric position. 4. Connect the wires to the power supply unit and apply 100 V until the blue dye front reaches the bottom of the gel. Disconnect the electrophoresis unit from the power supply, remove the lid and discard the running buffer. Remove the gel from plates with a spatula, discard the stacking gel, and wash the separated gel with distilled water to remove traces of running buffer. 5. Prepare the Coomassie brilliant blue G-250 staining 1 day before the staining process [23]. Place the gel in a tray containing 500 mL of staining solution. Incubate overnight the gel in the staining solution. Once the gel is stained, discard the staining solution and cover the gel with 0.1 M Tris–H3PO4. Then, shake for 1–3 min. Discard the solution and cover the gel with 25 % (v/v) of methanol. Then, shake for 1 min. Remove the methanol and wash the gel with 20 % (w/v) of ammonium sulfate for 24 h. 6. Images are digitized using a GS-800 Calibrated Densitometer (Bio-Rad, Hercules, USA) and analyzed with Quantity One software (Bio-Rad, Hercules, USA). 7. Excise the bands of interest from the gel, and place them in different tubes containing distilled water until their processing.
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3.5 Two-Dimensional Electrophoresis
Preliminary 2-DE experiments can be carried out with the MiniProtean 3 system (Bio-Rad, Hercules, USA), using 7 cm pH 3–10 linear gradient strips (Bio-Rad, Hercules, USA) and 13 % polyacrylamide gels, to examine the pI range where the proteins are concentrated. In our case, the most protein spots were located in a pI range between 5 and 8. 1. Prepare a mix with 300 μg of proteins in 250 mL of rehydration solution on 1.5 mL tube. Load the samples in each lane of the 17 cm strip holders. 2. Remove the protective cover from the surface of the IPG strips and slowly lower the IPG strip (gel slide down) onto the rehydration solution, without trapping air bubbles. Then cover the IPG strip with 1–2 mL of mineral oil and apply the plastic cover. 3. Apply a low voltage (50 V) during rehydration for 12 h at 20 °C for improving the entry of high molecular weight proteins [33]. 4. After active rehydration, start isoelectric focusing at 20 °C using the following parameters 250 V for 2 min, followed by 150 min linear gradient from 250 to 10,000 V, and finally focus on up to 40,000 V at 10,000 Vh. 5. After IEF, the strips are immediately reduced and alkylated according to [33]. IPG strips are equilibrated in two steps. Firstly, it is performed with 2 % (w/v) DTT in equilibration buffer I for 10 min in agitation at room temperature; secondly, it is carried out with 2.5 % (w/v) iodoacetamide in equilibration buffer II for 10 min in agitation at room temperature. 6. The second dimension is performed on 13 % polyacrylamide gels using the Protean Dodeca Cell (Bio-Rad, Hercules, USA). The gels can be run at 150 constant volts until the dye reaches the bottom of the gel. 7. The gels are stained employing the colloidal Coomassie method [23]. Soak the gel in a tray containing 50 mL of staining solution. Incubate overnight the gel in the staining solution. Once the gel is stained, discard the staining solution and cover the gel with 0.1 M Tris–H3PO4. Then, shake during 1–3 min. Discard the solution and cover the gel with 25 % (v/v) of methanol. Then shake during 1 min. Remove the methanol and wash the gel with 20 % (w/v) of ammonium sulfate for 24 h. 8. Images are digitized using a GS-800 Calibrated Densitometer (Bio-Rad, Hercules, USA), and then analyzed with PD-Quest software v8.1 (Bio-Rad, Hercules, USA).
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9. Select a good image as a reference with a clear and representative spot pattern and with a minimum distortion, and align each of the images to the chosen reference. Select between three prominent spots to manually assignment, and use the automatic vector tool to add additional vectors. 10. After automatic spot detection and matching, check manually the spots with edition tools for correct detection. 11. Set gel groups according to the experimental design and normalize spot volume intensity ratios for each spot. 12. List all the spots together with their normalized volume. 3.6 Phylogenetic and Statistical Analyses
Prior to statistical and phylogenetic analyses, the volume of pixels for each band or spot is normalized according to the total volume of bands detected (SDS-PAGE) or to the total volume of valid spots in each gel (2-DE), respectively. Then, they are logtransformed, following the recommendations described by Valledor and Jorrin [34]. A multivariate analysis is carried out on two steps: firstly, hierarchical clustering is performed to check the entire dataset, and the results are indicated in dendrograms using the cluster function of the software used; secondly, the dataset is analyzed by the use of Principal Component Analysis (PCA). The settings used for the PCA analysis are: co-variance matrix type, three principal components, onefold change, and 0.4 correlation threshold for clusters. PCA results are shown as a biplot. Since the employed statistical methods tend to classify population together, this information is necessary for studying the possible correlation between distances and geographical and climate parameters.
3.7 Protein Identification
Spots are manually excised with a scalpel. Protein identification was carried out according to the Proteomics Service protocols of the University of Córdoba. Gel plugs are digested with modified porcine trypsin (Sequencing grade; Promega), by using an automatic ProGest digestion station (Genomics Solution). The conditions are two detained steps for 30 min with 200 mM ammonium bicarbonate in 40 % (v/v) ACN at 37 °C; twice washed with 25 mM ammonium bicarbonate for 5 min and 25 mM ammonium bicarbonate in 50 % (v/v) ACN for 15 min respectively; dehydration with 100 % (v/v) ACN for 5 min and sample dried; hydration using 10 μL trypsin in a solution of 25 mM ammonium bicarbonate at a final concentration of 12.5 ng/μL for 10 min a room temperature, and the digestion is proceeded at 37 °C for 12 h. Subsequently, digestion is stopped by adding 10 μL of a solution of 0.5 % TFA in water.
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Tryptic peptides are purified in an automatic ProMS station (Genomic Solutions) by using a resin C18 microcolumn (ZipTip, Millipore), and they are eluted directly with a matrix solution (α-cyano hydroxycinnamic acid at a concentration of 5 mg/mL in 70 % (v/v) ACN/0.1 % (v/v) TFA) on MALDI plaque in 1 μL of final volume. After the cocrystallization on plaque, samples are analyzed by MALDI-TOF/TOF mass spectrometry to obtain the peptide mass fingerprinting (MS) in a 4800 Proteomics Analyzer (Applied Biosystems). The settings are: 800–4,000 m/z range, with an accelerating voltage of 20 kV, in reflection mode, with delayed extraction set to “on”, and an elapsed time of 120 ns. Spectra are internally calibrated with peptides from trypsin autolysis (M + H+ = 842.509, M + H+ = 2,211.104) with an m/z precision of ± 20 ppm. Most abundant peptide ions are subjected to MS/MS analysis, providing information that can be used to define the peptide sequence. A combined search (PMF and MS/MS) is performed with GPS ExplorerTM software v3.5 (Applied Biosystems) over nonredundant NCBI databases using the MASCOT search engine (Matrix Science Ltd., London; http://www.matrixscience.com). The database search utilized the following parameters: taxonomy restrictions to Viridiplantae, one missed cleavage sites, 100 ppm mass tolerance in MS and 0.5 Da for MS/MS data, cysteine carbamidomethylation as a fixed modification, and methionine oxidation as a variable modification. The confidence in the peptide mass fingerprinting matches (p < 0.05) is based on the MOWSE score, and confirmed by the accurate overlapping of the matched peptides with the major peaks of the mass spectrum.
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Final Remarks Taking into account our own experience, in this chapter we have shown the usefulness of a basic proteomic approach—based on SDS-PAGE and 2-DE analyses of protein extracts from mature seeds and pollen—for variability studies in Holm oak. These analyses have allowed the separation and grouping of the populations according to its acorn morphometry, location (northern and southern), and climate conditions (xeric, mesic, and intermediate), confirming previous results of our research group by using acorn morphometry and NIRS chemical composition [15, 16, 35]. Therefore, gel-based Proteomics (SDS-PAGE and 2-DE) can be used to detect variability between different populations from different environments, and to correlate it to environmental conditions (Fig. 1).
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Notes 1. Check the list of compatible chemicals and potential interfering chemicals typically found in the protein extraction buffer. 2. Do not freeze stock vial solutions more than once. 3. Prepare this solution on fresh each time. 4. TEMED accelerates the decomposition of APS molecules into sulfate free radicals and these, in turn, initiate the polymerization. 5. Prepare running buffer as 10× native buffer (0.25 M, Tris, 1.92 M glycine, and 1 % (w/v) SDS). Weigh 30.3 g of Tris, 144 g of glycine, and 10 g of SDS; mix and make it to 1 L with water. The pH of this solution should not be adjusted, and stored at room. 6. It is necessary to wear gloves and lab coat for all the procedures. 7. Be careful with liquid nitrogen due to its cool temperature (−195.8 °C). It could cause severe frostbite. Pay attention to laboratory safety regulation. 8. Be particularly careful when handling the reagents TCA and phenol (consult safety data sheets) because they are corrosive. Use the fume hood with volatile reagents. 9. Be careful in not throwing out the pellet. 10. The volume of the solubilization solution added will depend on the quantity of precipitated proteins. It is advisable that samples be well concentrated. 11. There are three phases, namely: the upper phase (which is the phenolic phase where the proteins are), a white interphase, and a lower aqueous phase. Try to not to take part of the white interphase.
Acknowledgments Jose Valero was recipient of an Alban Program fellowship (I06D00010MX). This work was supported by the Spanish Ministry of Science and Innovation cofinanced by the European Community FEDER funds: CGL2008-04503-C03-01/BOS, AGL2002-00530, and AGL2009-12243-C02-02; the Regional Government of Andalusia (Junta de Andalucía; the University of Córdoba (AGR-0164: Agricultural and Plant Biochemistry and Proteomics Research Group)); and the Autonomous University of Juarez City.
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