Chapter 14 Proteomic Analysis of Lung Tissue by DIGE Jarlath E. Nally and Simone Schuller Abstract Lungs perform an essential physiological function, mediated by a complex series of events that involve the coordination of multiple cell types to support not only gaseous exchange, but homeostasis and protection from infection. Guinea pigs are an important animal disease model for a number of infectious and noninfectious pulmonary conditions and the availability of a complete genome facilitates comprehensive analysis of tissues using the tools of proteomics. Here, we describe the application of 2-D Difference Gel Electrophoresis (DIGE) to compare, quantify, and identify differential protein expression of proteins in lung tissue from guinea pigs with leptospiral pulmonary hemorrhage syndrome (LPHS) compared to noninfected controls. 2-D DIGE is a powerful technique that provides novel insights into the dynamics of the complex lung proteome during health and disease. Key words DIGE, SDS-PAGE, Proteomics, Lung, Pulmonary, CyDye, Guinea pigs

1

Introduction The lungs, one of the largest organs of the human body, facilitate respiration. This physiological function is regulated by a complex series of events that involve the coordination of multiple cell types, including pneumocytes, bronchial epithelium, alveolar macrophages, endothelial cells, and interstitial cells, that not only supports gaseous exchange, but homeostasis and protection from infection. Transcriptomic analysis of lung tissue indicates that 73% of all human genes are expressed in the lung, of which 183 are expressed at elevated levels compared to other tissue types [1–3]. A comprehensive analysis of the complete proteome of the human lung is being performed (http://www.proteinatlas.org/ humanproteome/lung) [4]. Proteomic analysis of lung tissue is being used to provide novel insights into the mechanisms of many disease processes including infectious [5, 6] and noninfectious conditions [7–11]. Guinea pigs represent an important disease model for a number of infectious and noninfectious pulmonary conditions such as

Kay Ohlendieck (ed.), Difference Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1664, DOI 10.1007/978-1-4939-7268-5_14, © Springer Science+Business Media LLC 2018

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LPHS [12], tuberculosis [13], Legionnaires disease [14], allergic asthma [15], chronic bronchitis [16], and preterm respiratory distress syndrome [17]. Guinea pigs share a number of similarities with humans with regard to hormonal and immunologic responses [18], pulmonary physiology [19], and the corticosteroid response [20]. The immunological genes of guinea pigs are more similar to humans than that of the mouse [21]. This species therefore represents a particularly important model for the human immune system. While the Broad Institute originally sequenced the guinea pig genome to 2
coverage as part of the Mammalian Genome Project to annotate the human genome, the guinea pig genome has now been published to full (7
) coverage (https://www.broadinstitute. org/guinea-pig/guinea-pig-genome-project). In addition, low sequence coverage from two outbred guinea pig strains, one additional inbred strain, and a Peruvian guinea pig as part of a SNP discovery project are currently being added. These findings are freely accessible to researchers and have opened up important new avenues of research investigations using genomic, transcriptomic, and proteomic techniques in this species. Both gel-free and gel-based proteomics techniques have been successfully applied to examine the dynamics of the proteome of lung tissue and have demonstrated the power of both the approaches. Gel free “shot gun” techniques are considered fast and reliable, but gel-based techniques, and in particular 2-D differential gel electrophoresis (DIGE), while being more labor intensive, provides a visual control and analysis of the sample, allowing for the targeted identification of protein spots of interest and the differentiation of selected protein isoforms and their respective posttranslation modifications [22]. Gel-based proteomics relies principally on sodium dodecyl sulfate polyacrylamide-gel electrophoresis (GE) to separate complex protein samples using both one-dimensional (1D) and twodimensional (2D) separation techniques. 1D GE allows for the separation of proteins according to molecular mass. In contrast, 2D GE is based on the separation of proteins according to both isoelectric point (Isoelectric focusing) and molecular mass (GE) which results in highly resolved protein spots. While 2D GE provides a good overview of the molecular mass and isoelectric point of the majority of proteins in a complex sample, gel-to-gel variability can limit the ability to directly compare protein abundance between samples. This limitation has been overcome with the development of 2D DIGE, a technique that uses fluorescent labeling of protein samples prior to protein separation in the same gel [5, 23]. While different dye sets are commercially available, this article will focus on the use of the cyanine-based dyes (CyDyes): Cy2, Cy3, and Cy5. Protocols have been developed such that a restricted number (~5%) of lysine residues on each protein are labeled with CyDyes [24, 25]. This fluorescent minimal labeling

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approach [26] allows for the separation of proteins from two different samples, labeled with two different CyDyes, to be separated on the same gel, and thus minimizes gel-to-gel variability. It also allows for the inclusion of an internal standard to normalize spot intensities across multiple gels. Further, fluorescent dyes increase the sensitivity of GE, allowing for the detection of up to 0.2 fmol of protein over a broad linear range (ca 20,000-fold concentration range) for quantitation, permitting the precise measurement of a wide range of protein spot intensities [22]. The principle of 2D DIGE is simple: two protein samples, labeled with different fluorescent CyDyes, typically Cy3 and Cy5, are separated on a single gel which also contains an internal standard. The internal standard comprises an equal amount of proteins from all samples included in the study labeled with a third CyDye, typically Cy2 [27]. Proteins with similar characteristics labeled with different fluorescent dyes co-migrate to the same position on the gel. Protein spots can then be imaged using a fluorescence scanner, equipped with filters to pick up the frequencies of the individual fluorescent dyes, thus generating three images representative of samples labeled with Cy3 and Cy 5, as well as the internal standard labeled with Cy2. The same internal standard can be used to normalize the relative spot intensities of protein spots across multiple gels and thus compare the relative abundance of specific proteins in each sample of interest [28]. Differentially expressed protein spots can then be identified and quantified by software analysis, and selected for further analysis, e.g., identification by mass spectrometry. Here, we describe the application of 2D DIGE as used to compare the proteome of lung tissue from guinea pigs with leptospiral pulmonary hemorrhage syndrome (LPHS) to that of noninfected controls [5, 29]. All proteins identified in this experiment were used to generate a 2-D guinea pig lung proteome map (http://proteomics-portal.ucd.ie/). This provides a reference gel map, facilitating future gel-based proteomic studies on the lungs of guinea pigs.

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Materials

2.1 Reagents and Consumables

Use analytical grade reagents whenever possible. 1. Amidosulfobetaine-14 (ASB-14). 2. Acrylamide stock, available from various commercial sources (see Note 1). 3. Bind-silane (GE Healthcare). 4. CyDyes (Cy2, Cy3, Cy5); available from various commercial sources.

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5. Dimethylformamide (DMF, anhydrous). 6. Electrode pads (precut). 7. Fluorescent stain (SyproRuby®; Deep Purple™). 8. Immobilised pH gradient (IPG) strips (pH 3–10 NL, pH 4–7; 7 cm and 24 cm Immobiline DryStrips). 9. IPG strip cover fluid (GE Healthcare). 10. IPG buffer corresponding to the pH range of the IEF strips. 11. Lint free tissue wipes. 12. Low protein retention tubes. 13. pH indicator paper with a narrow pH range (e.g., 7.5–9.5). 2.2

Equipment

1. Sonicator. 2. DryStrip rehydration tray (GE Healthcare). 3. IPGphor IEF apparatus (GE Healthcare). 4. Low fluorescent glass plates 24 cm (GE Healthcare). 5. Stand for casting large gels including blank cassettes and separator sheets (GE Healthcare). 6. DaltSix electrophoresis and cooling unit (GE Healthcare). 7. Fluorescent gel imager (e.g., Typhoon 9400 Variable Mode Imager). 8. Image analysis software (Progenesis® same spots, DCyder, ImageQuant by GE Healthcare, ImageJ).

2.3 Preparation of Reagents

All the solutions are prepared with ultrapure/double-distilled water unless otherwise stated. 1. 1% Bromophenol blue stock solution: Mix 100 mg Bromophenol Blue and 60 mg Tris base. Bring up to 10 ml final volume. 2. Solubilization Buffer: 7 M Urea, 2 M Thiourea, and 1% ASB14; Mix 42.042 g of Urea, 15.22 g of Thiourea, and 1 g of ASB-14. Bring up to 100 ml with double distilled water. Make 1 ml aliquots and freeze at 20  C. 3. Dye solutions: Cy2, Cy3, and Cy5 dyes are commercially sourced and should be stored at 20  C until use. After centrifugation to ensure reagents are at the bottom of the tubes, dyes are reconstituted by adding fresh dimethylformamide (DFM) to obtain 1 mm stock solutions. The stock solutions are then further diluted with DFM to 400 pmol/μl working solutions. 4. Quenching solution: Prepare 1 μl of 10 mM lysine per μl of CyDye added. 5. Rehydration buffer: Solubilization buffer containing 30 mM DTT, 0.5% IPG buffer (0.5%), and 1 μl of Bromophenol blue stock solution. Use IPG buffer corresponding to the pH range

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of the IPG strip. Final volumes are dependent on strip length (125 μl for 7 cm and 450 μl for 24 cm strips). 6. 12% Polyacrylamide gels (makes six 24 cm gels): Mix 253 ml of double distilled water, 187.5 ml of 40% Acrylamide, 6 ml of 10% SDS, and 150 ml of Tris Buffer pH 8.8. Degas for 2 h using a vacuum pump, while mixing with a stir bar, then add 166 μl TEMED and 3.3 ml of 10% APS just before pouring the gel. Stock solutions of 10% APS can be prepared ahead of time and stored in aliquots at 20  C. 7. Equilibration buffer: 6 M urea, 75 mm Tris–HCl pH 8.8, 29.3% glycerol, 2% SDS, and 0.002% bromophenol blue. For 200 ml buffer, mix 72.1 g Urea, 6.7 ml of Tris–HCl ph 8.8, 69 ml of Glycerol, 4 g SDS, 400 μl Bromophenol blue stock solution and add double distilled water to 200 ml. Freeze 20 ml aliquots at -20  C. Add DTT or Iodoacetamide, to a final concentration of 1% or 2.5% respectively, just before use. Calculate 20 ml of Equilibration buffer and 0.2 g of DTT or 0.5 g of Iodoacetamide per strip. 8. 10
SDS Electrophoresis Buffer: Mix 60.5 g Tris base, 288.2 g Glycine, and 20 g SDS. Bring up to 2 l with double distilled water. To obtain 1
SDS running buffer dilute 1:9 with double distilled water. To obtain 2
SDS running buffer dilute 1:5 with double distilled water. You need about 4 l of 1
SDS running buffer and 1 l of 2
SDS running buffer to complete this experiment. 9. Agarose Sealing solution: Mix 0.5 g Agarose, 200 μl of 1% Bromophenol stock solution. Bring up to 100 ml with 1
Electrophoresis buffer. 10. Sypro Fixation buffer: Mix 500 ml methanol and 70 ml acetic acid. Bring up to 1 l with double distilled water. 11. Sypro Wash buffer: Mix 100 ml methanol and 70 ml acetic acid. Bring up to 1 l with double distilled water.

3

Methods

3.1 Experimental Design

When planning a 2D DIGE experiment, several factors including the number of biological replicates, sample randomization, and general time management should be considered [28]. 1. Number of biological replicates: The number of samples to be included in the experiment should be decided based on the expected biological variation across samples (e.g., lung tissues from a standardized laboratory animal model versus patient materials). The heterogeneity of samples can be estimated by performing preparatory 1D and/or 2D GE and comparing

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Table 1 Experimental design. Comparison of protein abundance between six biological replicates of disease (A1–A6) and control groups (B1–B6). Samples are labeled with either Cy3 or Cy5 using a “dye swap.” The pooled internal standard is labeled with Cy2. Samples are randomized to one of six gels

Gel

Cy2 Pooled internal standard

Cy3 50 μg in 400 pmol of Cy3

Cy5 50 μg in 400 pmol of Cy5

1

50 μg (4.17 μg each of samples A1–6 and B1–6)

B2

A5

2

50 μg (4.17 μg each of samples A1–6 and B1–6)

A1

B3

3

50 μg (4.17 μg each of samples A1–6 and B1–6)

A6

B1

4

50 μg (4.17 μg each of samples A1–6 and B1–6)

A3

B5

5

50 μg (4.17 μg each of samples A1–6 and B1–6)

B4

A2

6

50 μg (4.17 μg each of samples A1–6 and B1–6)

B6

A4

protein band/spot patterns. A minimum of three samples per group should be included in an experiment to allow for a meaningful statistical analysis. To limit run-to-run variability, all gels to be included in the analysis should be run in parallel either in one or more 6 or 12 gel appliances. 2. Sample randomization. In order to limit sample labeling bias, samples should be randomized so that equal numbers of both groups are labeled with Cy3 and Cy5 (dye swap). Cy2 is used to label the internal standard. Tonge et al. (2001) compared the variability of different CyDye combinations and found that a comparison of Cy3 and Cy5 labeled samples was less variable than other dye combinations, likely due to the fact that Cy2 is a slightly weaker fluorescent agent and therefore associated with a less favorable signal-to-noise ratio [25]. Karp and Lilley (2005) confirmed this observation. Labeled samples are then randomized to the gels in order to remove bias due to systematic errors caused by experimental artifact. An example for sample randomization on 6 gels is shown in Table 1. 3. Time management. The full experiment will take 3 days to complete. The workflow can be interrupted at various stages as indicated; however, careful planning is necessary to ensure vital equipment and facilities are available when they are needed. 3.2 Sample Preparation

1. Choice of sample. Careful consideration should be given to the choice of sample. Lung tissue is complex, containing a multitude of different structures and cell types, as well as blood and potentially oedema fluid. Because of the high sensitivity of DIGE to detect differences in protein abundance, structures

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of interest should be chosen carefully (e.g., non-hemorrhagic, non-oedematous areas; bronchi vs parenchyma) to avoid introduction of artifactual differences in protein abundance. Laser microdissection is a useful technique to capture specific areas of interest [30]. 2. Protein solubilization. Several methods can be used for sample preparation including grinding of frozen tissue, glass bead solubilization, and sonication. When using sonication, a good approach is to place 0.2 g of flash frozen lung tissues (flash frozen at the time of collection) in a 1.5 ml low protein retention Eppendorf tube containing 1 ml of solubilization buffer. The sample is sonicated in short bursts at maximum capacity, while being kept on ice to avoid overheating. The samples are then incubated overnight at room temperature. Samples are then centrifuged at 12,000
g for 5 min and the supernatant and pellet were separated, aliquoted, and frozen until further analysis. Whichever method is used, efficient solubilization of the majority of proteins should be examined by comparing protein band patterns of supernatant and pellet on a one dimensional gel (Fig. 1). 3. Protein quantification. To ensure correct labeling and equal loading, protein concentrations of the samples are quantified using an assay compatible with reducing and/or detergent agents, e.g., RC/DC protein assay kit; Bio-Rad. 4. Determination of spot resolution. As part of the preparatory work, the degree of protein spot resolution and spread over pH ranges for your samples should be determined via 2-D gel electrophoresis (Fig. 2). For this purpose, 25 μg of proteins can be loaded onto 7 cm IPG strips starting with a pH range of 3–10. After second dimension separation, the gels are inspected for spots at the extremes of this range and for overlapping of spots. The ideal pH range for your sample is the one that includes the majority of spots while providing enough resolution to minimize overlapping of individual protein spots (Fig. 2). Often a pH range of 4–7 adequately fulfils these requirements. Alternatively, 2D DIGE analysis can be performed on several pH ranges or the use of IPG strips with a nonlinear gradient, which provide higher resolution in the middle of the gel and lower resolution at the higher and lower end of the pH spectrum of the strip. When comparing lung tissue from guinea pigs with LPHS compared to noninfected controls by DIGE, over 1500 protein spots were aligned across all biological replicates; only 5 proteins spots were detectable as differentially expressed over a pH range of 3–10 compared to 130 proteins spots that were characterized as differentially expressed over a pH range of 4–7 due to improved protein resolution (data not shown) [5].

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Fig. 1 Optimization of sample preparation. Comparison of protein fractions from lung tissue of noninfected (a) and infected (b) guinea pigs after one or two sonication steps in solubilization buffer. Comparison of protein profiles between pellets and supernatants by 1D–SDS PAGE suggests that the majority of proteins are solubilized in supernatant 1. There were no significant differences in protein band patterns between solubilized proteins present in the supernatant after one (supernatant 1) or two (supernatant 2) sonication steps. Based on these results one sonication step was judged sufficient for solubilization of the majority of proteins from lung tissue for 2-D DIGE. Gels were stained with SyproRuby stain. Molecular mass markers are indicated

5. Check sample quality. Running of preparatory gels also provides the opportunity to screen for obvious differences in spot patterns between groups or technical problems with the sample, including streaking or lack of high molecular weight proteins indicative of protein sample degradation.

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Fig. 2 Determination of protein spot resolution and pH range of proteins from lung tissue of infected and noninfected guinea pigs via 2-D gel electrophoresis. Lung tissue from noninfected (a, c) and infected guinea pigs (b, d) were separated over pH 3–10 (a, b) or pH 4–7 (c, d). Images show good protein spot resolution. A number of protein spots were present outside the pH 4–7 range. Limited differences in protein spot patterns between infected and noninfected lung tissues are detected. Twenty-five μg of protein were loaded onto each gel. Gels were stained with SyproRuby stain. Molecular mass markers are indicated

3.3 Labeling Procedure

Powder-free gloves should be worn for all the procedures to avoid keratin contamination, which will interfere with mass spectrometry. 1. Samples are defrosted. 2. To optimize fluorescent labeling, each sample pH is adjusted to pH 8.5 with 50 mM NaOH (optimal labeling is between pH 8.0 and 9.0) (see Note 2). 3. The optimal protein concentration for labeling is 0.5–10 μg/μ l. To avoid pipetting errors, it is helpful to adjust all samples to the same protein concentration before going into the experiment. This considerably simplifies the task of combining samples for the internal standard and final sample mixes. 4. The internal standard is prepared by pooling equal amounts of sample from all samples included in the experiment (see Note 3).

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5. For each gel, 50 μg of protein from samples are labeled with 400 pmol of Cy3 or Cy5 (see Note 4). For each gel, 50 μg of protein of pooled internal standard is labeled with Cy2. The samples are left to incubate on ice for 30 min (see Note 5). The labeling is then quenched by the addition of 1 μl of 10 mM lysine per μl of CyDye added and samples are incubated for 10 min at room temperature. During the entire experiment, labeled samples are protected from light in order to limit degradation of the CyDye labels. After labeling the samples are combined as per experimental design. 3.4

Gel Preparation

As much of the quality of the final images relies on the quality of the gels, specific care should be given to producing high quality gels for DIGE. To ensure all the gels in the experiment have the same chemical and physical properties, a multi-gel caster allowing for the simultaneous pouring of all gels is necessary. 1. Low fluorescent plates are carefully cleaned (see Note 6) and front and back plates assembled. 2. The caster is prepared and filled, starting with a separator sheet and then by alternating plates and separator sheets until full. 3. Finish with a separator sheet (see Note 7). 4. The caster is then positioned upright on an absolutely level surface to ensure horizontal gel surfaces. 5. The gel matrix is then poured into the caster via a funnel avoiding the introduction of air bubbles (see Note 8). 6. The surface of the gels is then generously sprayed with 0.1% SDS solution. The gels should be given several hours for polymerization. They can be stored at 4  C in 2D running buffer for up to 4 days.

3.5 Rehydration of IPG Strips

1. Prepare rehydration solution. Pipette the required amount per strip in separate labeled tubes. Then add required amount of sample and solubilization buffer to add up to the desired volume (see Note 9). Again, this is simplified if all samples have been standardized to the same protein content. Carefully mix by pipetting up and down. Briefly spin down samples to remove all bubbles. 2. Remove IPG Strips from freezer. 3. Prepare rehydration tray. Make sure it stands level. 4. Carefully pipette final rehydration solution into wells. There should be no bubbles. Burst bubbles with a needle before placing the IPG strip with the gel side facing down onto the rehydration solution. Be careful to record strip numbers associated with each sample. Carefully cover strips with cover fluid

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to avoid desiccation. Cover the tray with lid, then put light excluding cover over the tray. Allow IPG strips to rehydrate for 10–20 h at room temperature. 3.6

2D SDS-PAGE

1. Isoelectric focusing. Isoelectric focusing can be performed using an Ettan IPGphor IEF System (GE Healthcare). The strip holder is positioned in the machine and all channels evenly covered with 108 ml of Immobiline DryStrip cover fluid. IPG Strips are placed, face up in the tray with the anodic (þ) end of the strip resting on the appropriate mark on the tray. Precut electrode pads are wet with 150 μl of deionized water and blotted until they are almost dry. The pads are placed on the ends of the IPG strips. The electrode assembly is placed over the top of all pads and locked (see Note 10) and the cover of the Ettan IPGphor is closed. Ensure that samples are run in the dark. Perform Isoelectric focusing according to the length and pH range of selected IPG strips. For 24 cm pH 4–7 strips, 3500 V for 75,000 VH (step 1), a gradient to 8000 V for 10 min (step 2), followed by 8000 V for 1 h (step 3), and 100 V for 5 h (holding step) work well. After isoelectric focusing strips can be frozen at 80  C for 2–4 days. 2. Strip equilibration. Strips are transferred into equilibration buffer with added 1% DTT and incubated on a shaker for 10 min. After a brief rinse with water, the strips are transferred into the second equilibration buffer containing 2.5% iodoacetamide for 10 min. Placing the IPG strip, with the gel side facing inward, in a Petri dish works well for this procedure. 3. SDS-PAGE. The strips are rinsed with electrophoresis running buffer and overlaid on 12% acrylamide gels, which are prepared upright in a stand (see Note 11). Agarose gel with bromophenol blue (tracking dye) is used to seal the strips (see Note 12). The gels are run using a DaltSix electrophoresis unit (GE Healthcare). Prepare the tank by inserting the anode assembly unit. Fill the unit with 1
SDS Electrophoresis buffer and turn the pump on. Turn on the cooler system (15  C). Insert the prepared gels into the unit. Fill the unused spaces with blank cassettes. Add more 1
SDS Electrophoresis buffer until the buffer is at or just below the “LBC (lower buffer chamber) start fill" line. Slide on the UBC (upper buffer chamber). Fill the upper chamber with 2
SDS Electrophoresis buffer to between the fill lines (approximately 0.8 l). Fill the LBC with 1
SDS Electrophoresis buffer to the same level as the upper chamber (approximately 4 l in total). Put the lid on the unit. Start the run (see Note 13). At the end of the run, when the dye front has just migrated off the end of the gel, switch off the power pack, disconnect and remove gels.

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1. Scanning. Gels can be scanned inside the glass plates. Plates should be thoroughly cleaned before scanning because high fluorescent specs might artificially increase the upper end of the dynamic range thus preventing the detection of low intensity spots. A Typhoon™ variable mode imager (GE Healthcare) is commonly used for gel scanning. This scanner has band pass filters to image each of the three CyDyes (520 nm for Cy2, 580 nm for Cy3, and 670 nm for Cy5). The gels are prescanned at a pixel size of 500 μm and the photomultiplier tube (PMT) voltage adjusted in order to ensure that the most intense spot on the gel is at the upper end of the dynamic range, thus ensuring that the full dynamic range of the detector is used. For the final scans, gels are scanned at a pixel size of 100 μm.

3.7 Image Acquisition and Analysis

2. Principle component analysis (PCA) is a multivariate statistical analysis technique, which allows for easy detection of outlying gels and visualization of clustering of results of the biological replicates according to their group. PCA is performed by comparing spot with significant differences in normalized spot volumes between the groups present on all gels. It provides a global perspective over the experimental variation, thus giving an idea whether the variation seen is due to biologic variation according to the grouping, or technical/random biological noise in the system. An example for good clustering of biological replicates into groups is shown in Fig. 3. 3. Spot analysis. Differences in spot volumes can be analyzed using dedicated software packages such as SameSpots (TotalLab) or DeCyder (GE Healthcare), PDQuest (Bio-Rad), Delta2D (Decodon). Freeware programmes such as ImageJ or QuickTime can also be used for image visualization and annotation. Principal Component Analysis

Principal Component 2

0.8 0.6 0.4 0.2 0.0 –0.2 –0.4 –0.6 –0.8 –0.8

–0.6

–0.4

–0.2

0.0

0.2

0.4

0.6

0.8

Principal Component 1

Fig. 3 Principle component analysis biplot of 2-D DIGE pH 4–7. Principle component analysis (PCA) allows for easy detection of outlying results and visualization of clustering of results of the six biological replicates according to their group [5]. The biplot shows excellent clustering of expression levels of significantly differently expressed spots on the six gels according to the relevant group (LPHS pink; control blue)

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Fig. 4 Three different ways to illustrate differences in spot volumes using spot 695 from the pH 4–7 experiment [5]. (A) 2-D image of protein spot 695 from 12 biological replicates. (B) 3-D image of mean spot volumes of spot 695 in LPHS and control samples; (C) Graphic illustration of spot volumes for spot 695 in LPHS and control groups. The spot intensities (A) and volumes (B, C) of protein spot 695 are significantly higher in LPHS lung tissue compared to controls. This protein was identified by mass spectrometry as alpha-1antiproteinase S precursor, an acute phase protein [5]

Gel images are inspected and imported into the selected programme. Prior to analysis, images are first cropped (making sure high signal areas at the borders of the gels and spot picking reference tags are removed), then the samples on each gel assigned to the respective experimental groups (see Note 14). Spots are then aligned by the programme and the spot volumes of the individual samples normalized against the internal standard. Spot volumes of protein spots aligned across all gels are then compared between groups. Statistical criteria for significant differences in spot volumes typically are set at p < 0.05, power > 0.8, and q < 0.01 (false discovery rate). While the software will do most of this work automatically, visual inspection of single protein spots of interest is advised before assembling the final list of protein spots for picking. Figure 4 illustrates the analysis of a protein spot with significant

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differences in abundance in lung tissue from experimentally infected guinea pigs (LPHS) compared to the noninfected control group [5]. 3.8 Generation of a Master Gel and Spot Picking

For protein identification, protein spots can be picked from the gels included in the 2D DIGE analysis, or alternatively from separately run master gels. The advantage of using master gels is that greater amounts of protein can be loaded. Typically, 500–1000 μg of protein are loaded per master gel. 1. Preparation of master gels for spot picking. Before pouring the gel for second dimension separation, the front glass plate should be treated with a solution containing 80% ethanol, 2% acetic acid, and 0.1% Bind-silane (GE Healthcare) in order to immobilize the gel on the front plate during staining and spot picking. Once dry, a reference marker is attached on the midpoint of the left and right side margins. These markers served as coordinates and reference points when using an automated spot picker. 2. Protein separation. Proteins are separated using the same protocol as described for DIGE. 3. Fixation and staining. After second dimension protein separation, the two glass plates are opened and the gel that is immobilized to the front plate, is first placed in fixation solution (50% methanol and 7% acetic acid) for 1 h and then stained with SyproRuby stain (Invitrogen) overnight. After staining, the gel is destained using 10% methanol and 7% acetic acid for 1 h to reduce background. If the gel is to be scanned again using the Typhoon™ variable mode imager, the glass plates are reassembled, and scanned with the front plate facing up, as previously described. A spot picking list with the coordinates of all selected spots can be produced using SameSpots (TotalLab). Because gels are scanned with the front plate on the top of the gel, and automated spot pickers typically work with the front plate under the gel, the X coordinate has to be corrected to account for the inversed image using the formula Xcorrected ¼ Xmax-x (X being the corrected coordinate, Xmax being the width of the image in pixels, and x being the previous coordinate). This step is obviously not necessary if a combined scanner-spot picker robot is used.

4

Notes 1. Acrylamide is a neurotoxin. It is important to wear appropriate personal protective equipment and use appropriate handling precautions.

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2. To test sample pH, pipette one droplet of sample and standard on pH indicator paper. If the pH is below 8 the CyDye will not bind, and if it is above 9, multiple dye molecules can bind to the protein, or to different amino acids, which will be negatively charged at high pH. 3. Make sure you have enough standard for all gels included in the experiment, plus extra for potential repeats and pipetting losses. 4. The protein to dye ratio recommended by the manufacturer is 400 pmol dye for 50 μg protein. It is possible to work with 25 μg protein and 200 pmol/dye without loss of sensitivity in case of very small samples or to reduce costs for the CyDye. 5. To synchronize the labeling time, the dye can be applied to the inner wall of the sample tube. The tubes are then all spun together, vortexed and spun again in a microcentrifuge to mix the dye well with the sample. 6. As gels are scanned between the glass plates, these have to be absolutely free of stain or dust. First wash the plates with water, and use ethanol and lint-free tissue to carefully clean the plates before casting the gels. 7. To avoid leakage, remove the gray foam seal from the groove in the faceplate of the caster and lubricate with Vaseline to help ensure a liquid tight seal and then put back in place. Be careful to also obtain a good seal between the bottom of the plates and the casting stand to avoid leakage of the gel matrix. 8. Gentle tapping of the caster stand from both sides with styrofoam blocks allows for trapped air to be released to the surface after pouring. 9. Some authors advise to not include the protein samples in the rehydration solution. Cup loading is used instead. 10. When using 24 cm strips, the top and bottom electrode units have to be placed with the electrodes facing away from each other in order to accommodate the length of the strips. 11. By convention the strips are placed with the acidic end to the left of the gel. Position the strips with the plastic backing against the inside of the back plate. The gel surface of the strip should not be touching the front plate. Then gently push the strip down until the entire lower edge of the strip is in contact with the top surface of the gel. 12. Allow for enough time for the agarose gel to cool down and solidify before moving the gels. 13. You can set up the power pack to 4 W/ gel for 1 h followed by 17 W/ gel until the bromophenol blue gets out of the gels (approx. 4:30 min). However, it works well to run the gels at

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0.5 W/gel for 1 h and then at 2 W/gel overnight. The power is increased the following morning to 17 W/gel until the tracking dye reaches the bottom edge of the gel. This allows for enough time to perform the scanning of the gels in one go on day 3 of the experiment. 14. After scanning of the gels, crop areas with spacers, IPG strips, and other areas, which might show autofluorescence and therefore interfere with the analysis.

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