Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

CHAPTER

Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

6 Jonathan S. Minden

Department of Biological Sciences, Carnegie Mellon University, USA

CHAPTER OUTLINE 1  Purpose��������������������������������������������������������������������������������������������������������������� 112 2  Theory����������������������������������������������������������������������������������������������������������������� 112 3  Equipment����������������������������������������������������������������������������������������������������������� 115 4  Materials������������������������������������������������������������������������������������������������������������� 116 4.1  Solutions and Buffers—Step 1������������������������������������������������������������� 117 4.2  Solutions and Buffers—Step 2������������������������������������������������������������� 117 4.3  Solutions and Buffers—Step 3������������������������������������������������������������� 118 4.4  Solutions and Buffers—Step 4A����������������������������������������������������������� 119 4.5  Solutions and Buffers—Step 4B����������������������������������������������������������� 119 4.6  Solutions and Buffers—Step 6������������������������������������������������������������� 120 4.7  Solutions and Buffers—Step 7������������������������������������������������������������� 120 4.8  Solutions and Buffers—Step 10����������������������������������������������������������� 121 5  Protocol�������������������������������������������������������������������������������������������������������������� 121 5.1  Step 1: Casting Second-Dimension Gel������������������������������������������������� 122 5.2  Step 2: IEF Strip Rehydration�������������������������������������������������������������� 123 5.3  Step 3—Sample Preparation���������������������������������������������������������������� 125 5.4  Step 4A—Minimal Protein Labeling with Lysine-Reactive Dyes���������������� 126 5.5  Step 4B Saturation Protein Labeling with Cysteine-Reactive Dyes������������ 127 5.6  Step 5—First-Dimension Electrophoresis: IEF���������������������������������������� 129 5.7  Step 6—Equilibration�������������������������������������������������������������������������� 132 5.8  Step 7—Second-Dimension Electrophoresis������������������������������������������ 132 5.9  Step 8—Image Acquisition������������������������������������������������������������������ 135 5.10  Step 9—Image Analysis��������������������������������������������������������������������� 135 5.11  Step 10—Spot Picking���������������������������������������������������������������������� 137 5.12  Step 11—Protein Identification���������������������������������������������������������� 138 Methods in Cell Biology, Vol 112 Copyright © 2012 Elsevier Inc. All rights reserved.

0091-679X http://dx.doi.org/10.1016/B978-0-12-405914-6.00006-8

111

112

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Abstract Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as “spots” with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, DIGE is capable of reliably detecting as little as 0.2 fmol of protein, and protein differences down to ±15%, over an approximately 10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2–3 days to complete.

1  PURPOSE This protocol describes a two-dimensional electrophoresis (2DE) gel method for detecting protein differences between two or three samples of complex mixtures of intact proteins, where proteins may differ in abundance, alternative splicing or ­posttranslational modification.

2  THEORY The central goals of proteomics include identifying protein changes that differentiate normal and diseased states in cells, tissues or organisms and examining how protein changes correlate with developmental age and environment. The first stage in comparative proteomics is to separate complex mixtures of protein into individual components; this is typically done using gel electrophoresis (at the whole protein level) or column chromatography (at the peptide level). Both these separation schemes have advantages and disadvantages. We have focused on 2DE because of its accessibility to most laboratories. This approach was described simultaneously by several groups in 1975 (Klose, 1975; O’Farrell, 1975; Scheele, 1975). Despite the substantial advances in the technology since its launch—the most notable of which was the introduction of immobilized pH gradients in the first dimension (Gorg, Postel, & Gunther, 1988; Hamdan & Righetti, 2005)—some of the more significant systemic shortcomings have remained unsolved. The most troublesome of these is the inherent lack of reproducibility between gels. Efforts to surmount this limitation have mostly focused on developing computational methods for gel matching. These approaches have had limited success because the sources of gel-to-gel variation are numerous, complex and difficult to model. Difference gel electrophoresis (DIGE) was developed to overcome the irreproducibility problem in the 2DE methodology by labeling two samples each with a

2  Theory

different fluorescent dye before running them on the same gel (Fig. 1) (Ünlü, Morgan, & Minden, 1997; Viswanathan, Ünlü, & Minden, 2006). The fluorescent dyes originally used in DIGE, Cy3-NHS and Cy5-NHS (Fig. 2A), are cyanine based, molecular-weight matched, amine reactive and positively charged. These

FIGURE 1 Schematic representation of DIGE analysis. Extracts are made of two cell samples, denoted “A” and “B.” These extracts are separately labeled with Cy3-NHS and Cy5-NHS, which covalently link to lysine residues. A low stoichiometry of labeling is used, where approximately 5% of all proteins carry a single dye molecule. The labeled protein extracts are then combined and coelectrophoresed on a 2DE gel. The gel is then imaged on a fluorescent gel imager at the Cy3 and Cy5 wavelengths. Shown here is a color overlay of Cy3 (green) and Cy5 (red) images of Drosophila embryo extracts. Regions of equal Cy3 and Cy5 signals appear yellow. MWt, molecular weight. See the color plate.

113

114

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

characteristics, coupled with substoichiometric labeling, result in no electrophoretic mobility shifts arising between the two differentially labeled samples when they are coelectrophoresed. Therefore, in DIGE, every individual protein isoform in one sample superimposes with its differentially labeled counterpart in the other sample, allowing for more reproducible and facile detection of differences. The ratio of fluorescence of commonly expressed proteins is constant, whereas differently expressed or modified proteins display a higher fluorescent signal of one fluorophore versus the other. DIGE is a sensitive technique, capable of detecting as little as 0.2 fmol of protein, and this detection system is linear over an approximately 10,000-fold concentration range (Gong et al., 2004; Lilley & Friedman, 2004; Ünlü et al., 1997). The minimal labeling cyanine dyes, Cy3-NHS and Cy5-NHS, are suitable for samples containing 100–200 μg of protein. For very precious samples containing 1–3 μg of protein, such as those isolated from microscopic tissue samples or tissue obtained by laser capture microdissection, saturation labeling dyes have been developed (Sitek et al., 2006). These dyes, Cy3-Mal and Cy5-Mal, are cysteine-reactive dyes that couple Cy3 and Cy5 to proteins via a maleimide reactive group (Fig. 2B). The most important considerations in performing DIGE experiments are experimental design and sample preparation (Shaw & Riederer, 2003). DIGE has been used to

FIGURE 2 Chemical structure of DIGE dyes. (A) Minimal labeling dyes, propyl-Cy3-NHS and methyl-Cy5-NHS are shown here. These compounds are charge and mass matched. The overall charge of these dyes approximates that of lysine. The fluorescent characteristics of Cy3 and Cy5 are dictated by the three-carbon and five-carbon polyene chains, respectively, linking the two indoline rings. (B) Saturation labeling dyes, propyl-Cy3-Mal and methyl-Cy5-Mal are shown here. These compounds are charge and mass matched. The overall charge of these zwitterionic dyes matches that of cysteine.

3  Equipment

analyze proteome changes from a wide variety of cell types and body fluids, including serum (Lilley, 2002; Marouga, David, & Hawkins, 2005; Okano et  al., 2006; Wang et al., 2005). The sample preparation protocol depends on the cell type. Most samples require mild homogenization in lysis buffer to extract protein. DIGE is an extremely sensitive method, in which a 15% change in protein abundance is more than 2 SDs above the normal variation (Gong et al., 2004). One must take great care in deciding which samples to compare, while bearing in mind the sources of variation. If specific tissues are to be compared, one must carefully dissect the tissue to avoid variation. Hypothetically, if one has 10% contamination of neighboring tissue in one sample and 5% variation in another, it might lead to artifactual protein differences. One way to avoid tissue contamination is to use laser microdissection, which provides a precise method for capturing specific populations of cells (Kondo et al., 2003; Sitek et al., 2006; Zhou et al., 2002). Another source of variation can arise during sample cleanup or fractionation. If sample cleanup or fractionation is planned, it is best to label the samples independently and then combine them before cleanup or fractionation. This will alleviate variation due to handling. Simply measuring total protein after sample cleanup or fractionation will not guard against variation in loss of specific proteins during processing.

3  EQUIPMENT Microcentrifuge tubes(1.5 mL) Fitted pestle Microcentrifuge Vortex mixer Magnetic stirrer Stirring magnets (assorted sizes) Syringe 26-gauge needles Micropipettors Micropipette tips Pipettes Pipette bulb or pump Ice bucket Temperature-controlled heating block for microcentrifuge tubes Isoelectric focusing (IEF) strip rehydration tray Paper wicks Sample loading cups IEF strip manifold or individual strip holders IEF electrodes IEF apparatus Equilibration dishes or tubes Rotating mixer Whatman 3 MM chromatography paper

115

116

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Fine scissors Fine forceps Plastic, screw-cap centrifuge tubes (15 and 50 mL) 100-mm plastic Petri dishes 60-mm plastic Petri dishes Electrophoresis glass plates Gel casting stand Gradient maker Large-format vertical gel electrophoresis apparatus Power supply Graduated cylinders (100 mL and 1 L) Glass tray Paper towels Aluminum foil Plastic wrap Platform rotator Fluorescent gel imager Image-analysis software Spot picking robot In-gel protein digestion robot (e.g. ProGest Digestion Robot, Genomic Solutions)

4  MATERIALS The source and purity of ingredients is critical, especially for solutions used for protein labeling and IEF. Double-distilled H2O Urea Thiourea CHAPS detergent Dithiothreitol (DTT) Tris(2-carboxyethyl)phosphate hydrochloride (TCEP) SDS: 20% w/v stock solution Tris Tris–HCl solution: 0.5 M, pH 6.8 Tris–HCl solution: 1.5 M, pH 8.8 Glycine Sucrose HEPES Sodium hydroxide (NaOH) Cy3-NHS (e.g. GE Healthcare) Cy5-NHS (e.g. GE Healthcare) Cy3-Maleimide (Cy3-Mal) (e.g. GE Healthcare)

4  Materials

Cy5-Maleimide (Cy5-Mal) (e.g. GE Healthcare) Iodoacetamide Acrylamide: 30% T, 2.6% C stock solution N,N,N′,N′-tetramethylethylenediamine (TEMED) Ammonium persulfate (APS) IEF strips (e.g. pH 3–10 nonlinear (NL), 18 cm, GE Healthcare) Gel covering fluid (light mineral oil) Dimethyl formamide (DMF) (anhydrous, 99.9%) Methylamine Hydrochloric acid (HCl) Bromophenol blue Acetic acid (glacial) IPG buffer (e.g. GE Healthcare) Glycerol (spectral or pesticide grade) Agarose: standard low Mr Methanol (HPLC grade) n-Butanol Bradford reagent IEF strip-cleaning detergent (e.g. GE Healthcare) RBS 35 detergent (concentrate, e.g. Pierce) Ethanol (absolute)

4.1  Solutions and Buffers—Step 1 Component

Final concentration

Amount

10% ammonium persulfate (APS) 1 g

Ammonium persulfate 10% Add water to 10 mL. Store as 1 mL aliquots at −20 °C. Water saturated n-butanol

100% 150 mL n-butanol Add 50–75 mL water. Shake vigorously and allow it to separate with the n-butanol layer on top.

4.2  Solutions and Buffers—Step 2 Component

Final concentration

Stock

Amount/L

Bromophenol blue solution Bromophenol blue 1% Tris–HCl pH 8.8 50 mM Add water to 10 mL. Store at room temperature.

1.5 M

100 mg 0.33 mL

117

118

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Component

Final concentration

Stock

Amount/L

17.4 M 1%

2.1 g 0.76 g 0.2 g 0.014 g 0.6 μL 10 μL

Rehydration buffer Urea 7 M Thiourea 2 M CHAPS 4% DTT 10 mM Acetic acid 2 mM Bromophenol blue 0.002% Add water to 5 mL. Store as 0.5-mL aliquots at −80 °C.

4.3  Solutions and Buffers—Step 3 Component

Final concentration

Stock

Amount/L

HEPES pH 8.0 buffer 1 M 119.16 g HEPES NaOH 10 N to pH 8.0 Add water to 400 mL. While stirring over magnetic stirrer, add NaOH dropwise to make pH 8.0. Add water to bring the final volume to 500 mL. Filter sterilize. Store at room temperature. Cell lysis buffer with DTT 7 M 3.36 g Urea Thiourea 2 M 1.22 g CHAPS 4% 0.32 g DTT 10 mM 0.0123 g HEPES pH 8.0 10 mM 1 M 80 μL Weigh dry components into a 15-ml screw-cap tube. Add water to 7 mL. Rotate gently end over end until the solids are dissolved. Do not heat above room temperature. Once dissolved, add HEPES buffer and water to bring the volume to 8 mL. Store as 0.5-mL aliquots at −80 °C. Cell lysis buffer without DTT 7 M 3.36 g Urea Thiourea 2 M 1.22 g CHAPS 4% 0.32 g HEPES pH 8.0 10 mM 1 M 80 μL Weigh dry components into a 15-mL screw-cap tube. Add water to 7 mL. Rotate gently end over end until the solids are dissolved. Do not heat above room temperature. Once dissolved, add HEPES buffer and water to bring the volume to 8 mL. Store as 0.5-mL aliquots at −80 °C.

4  Materials

4.4  Solutions and Buffers—Step 4A Component

Final concentration

Stock

Amount/L

Cy3-NHS dye solution 1 mM 5 nmol vial Cy3-NHS DMF (dry) 5 μL Allow the Cy3-NHS vial to warm to room temperature. Add DMF. Vortex briefly and then spin briefly in a microcentrifuge. Repeat vortex and centrifugation to ensure complete dissolution of the dye. Store at −80 °C. Cy5-NHS dye solution 0.83 mM 5 nmol vial Cy5-NHS DMF (dry) 6 μL Allow the Cy5-NHS vial to warm to room temperature. Add DMF. Vortex briefly and then spin briefly in a microcentrifuge. Repeat vortex and centrifugation to ensure complete dissolution of the dye. Store at −80 °C. Quenching solution 5 M 40% 38.8 ml Methyl amine HEPES 10 mM 11.6 M 2.38 g HCl To pH 8.0 ∼60 mL Dissolve HEPES in methyl amine solution. In a fume hood with the solution stirred on ice, slowly add HCl until the pH reaches 8.0. Add water to 100 mL. Store as 1-mL aliquots at −80 °C.

4.5  Solutions and Buffers—Step 4B Component

Final concentration

Stock

Amount/L

TCEP solution 10 mM ∼2.9 mg TCEP Weigh approximately 2.9 mg TCEP. Add water to make a 2.87 mg/mL solution. Dissolve and store as 0.1-mL aliquots at −80 °C. Cy3-Mal dye solution 10 mM 300 nmol vial Cy3-Mal DMF (dry) 30 μL Allow the Cy3-Mal vial to warm to room temperature. Add DMF. Vortex briefly and then spin briefly in a microcentrifuge. Repeat vortex and centrifugation to ensure complete dissolution of the dye. Store at −80 °C.

119

120

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Component

Final concentration

Stock

Amount/L

Cy5-Mal dye solution Cy5-Mal 10 mM 300 nmol vial DMF (dry) 30 μl Allow the Cy5-Mal vial to warm to room temperature. Add DMF. Vortex briefly and then spin briefly in a microcentrifuge. Repeat vortex and centrifugation to ensure complete dissolution of the dye. Store at −80 °C. 7M DTT solution 7 M ∼108 mg DTT Weight approximately 108 mg TCEP. Add water to make a 1.08 g/mL solution (∼100 μL). Dissolve and store as 0.1-mL aliquots at −80 °C.

4.6  Solutions and Buffers—Step 6 Component

Final concentration

Stock

Amount/L

Equilibration buffer 50 mM 1.5 M 6.7 mL Tris–HCl pH8.8 Urea 6 M 72 g Glycerol 30% 60 mL SDS 2% 20% 20 Bromophenol blue 0.002% 1% 0.4 mL In a 250-mL beaker containing 72 g of urea, add water to approximately 100 mL. Stir gently at room temperature until the urea is dissolved. Add the rest of the components, transfer to a 250-mL graduated cylinder and add water to 200 mL. Store at 4 °C for up to 6 weeks. Equilibration dishes For each IEF strip, construct an equilibration dish by fusing the lid from a 60-mm plastic Petri dish to the center of a 100-mm plastic Petri dish. Place two to three drops of acetone on the top of a 60-mm plastic Petri dish lid, invert it and press it into the center of a 100-mm plastic Petri dish, forming a 20-mm wide channel in the 100-mm Petri dish. Retain the 100-mm Petri dish lid.

4.7  Solutions and Buffers—Step 7 Component

Final concentration

Stock

Amount/L

0.5 M 1%

0.5 g 10 mL 0.1 mL

Agarose-sealing solution Agarose Tris–HCl pH 6.8 Bromophenol blue

1% 125 mM 0.002%

5  Protocol

Component

Final concentration

Stock

Amount/L

0.25 mL SDS 0.1% 20% Combine agarose, Tris–HCl, bromophenol blue and water to 44.75 mL. Heat to gentle boiling in microwave to dissolve the agarose. Once dissolved add SDS. Store as 5-mL aliquots at 4 °C. Take care to prevent boiling over when melting the gel by storing in oversized tubes. Running buffer 25 mM 12.11 g Tris Glycine 192 mM 57.6 g SDS 0.1% 20% 20 mL Dissolve Tris and glycine in 3.5 L of water. Add SDS solution. Add water to bring final volume to 4 L. Fixative solution 800 mL 200 mL

Methanol 40% Acetic acid 10% Add 1000 mL of water to obtain 2 L final volume.

4.8  Solutions and Buffers—Step 10 Component

Final concentration

Stock

Amount/L

1% acetic acid solution Acetic acid 1% Add acetic acid to 495 mL water. Store at room temperature.

5 mL

5  PROTOCOL Duration

Preparation

Tip

Time Preparation 1 day Protocol 2–5 days Each gel is loaded with 100–200 μg of each sample to be compared. We typically run two gels for each comparison where the samples are reciprocally labeled. This allows one to detect rare dye-dependent protein changes. The most critical aspect of 2D DIGE is experimental design and sample preparation. Variation in sample preparation may lead to artifactual differences. This is particularly true for dissected tissue and subfractionated samples.

See Fig. 3 for the flowchart of the complete protocol.

121

122

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Preparation

Isolation of proteome samples to be compared Preparation of required solutions and buffers

Step 1

Casting second-dimension gel

Step 2

IEF strip rehydration

Step 3

Sample preparation

Step 4A

Minimal protein labeling with lysine-reactive dyes

Step 4B

Saturation protein labeling with cysteine-reactive dyes

Step 5

First-dimension electrophoresis: IEF

Step 6

Equilibration

Step 7

Second-dimension electrophoresis

Step 8

Image acquisition

Step 9

Image analysis

Step 10

Spot picking

Step 11

Protein identification

FIGURE 3 Flowchart of the complete protocol, including preparation.

5.1  Step 1: Casting Second-Dimension Gel Overview

Duration 1.1

1.2

1.3 1.4

The second-dimension gels should be poured (or purchased) in advance. It is important to transition the IEF strip between the first- and seconddimension electrophoretic steps to avoid diffusion of protein spots. About 1 h contact time; 1 or more hours of curing time. Assemble the gel cassette using cleaned and dried plates. We use homemade 10–15% gradient gels. Use glass plates for large-format (approximately 20 × 20 cm) gels with 1.5-mm thick spacers. Set up a standard two-chamber gradient maker so that the outlet tube directs the acrylamide solution into the top of the gel cassette. Make sure the gradient mixer outlet is clamped off and the channel between the gradient mixer chambers is closed. Position the magnetic stirrer under the front chamber (the one closest to the mixer outlet) and set the stirrer to a medium stir rate. Mix acrylamide solutions according to Table 1. Just before pouring, add the APS and TEMED and mix by gently inverting the tubes for about 10 s.

5  Protocol

1.5 1.6 1.7 1.8 Caution Tip

Tip Tip

Tip

Tip

Tip

Simultaneously pour the heavy solution into the front chamber of the gradient maker and the light solution into back chamber. Open the mixing channel and the outlet stopcocks simultaneously to start pouring the gel. Once the entire gel is poured, overlay with 1 mL water-saturated n-butanol (top layer). Allow the gel to polymerize for ≥1 h Acrylamide is a neurotoxin; wear gloves and use appropriate handling precautions. Wear gloves at all times to avoid contact with acrylamide solutions and to prevent keratin contamination, which is a key obstacle to further analysis by MS. This step can be bypassed by purchasing premade gradient gels for 2DE, provided they do not contain any fluorescent contaminants. If one intends cutting protein spots for MS, the gels should be allowed to polymerize for ≥8 h. Unpolymerized acrylamide causes side-chain and amino-terminal modification of proteins that might pose problems for MS analysis. The gels should be layered with sufficient water-saturated n-butanol that the gels do not dry out. Make sure the opening of the outlet tube of the gradient maker is sufficiently wide such that the pouring process takes <2–3 min, otherwise the gel might begin to polymerize during the pouring process. We use a disposable 200-μL pipette tip cut at an angle, which fits snuggly between the glass plates and allows for rapid casting of the gradient gel. If pouring multiple gradient gels, rinse the gradient maker with water to flush out unpolymerized acrylamide. Make sure the water runs smoothly through the gradient mixer. Remove any polymerized acrylamide before pouring the next gel. Working quickly avoids clogs due to premature polymerization.

See Fig. 4 for the flowchart of Step 1.

5.2  Step 2: IEF Strip Rehydration Overview Duration 2.1 Tip Tip Tip Tip Tip

IEF strips come in a dried state. They must be rehydrated in the appropriate solution. Less than 30 min contact time; 10–20 h rehydration time. Rehydrate IEF strips in a rehydration tray according to the manufacturer’s instructions. Make sure to add the appropriate pH range IPG buffer to the rehydration buffer to a 2% final concentration. Make sure to add the exact volume of rehydration buffer as recommended. Avoid the inclusion of air bubbles under the IEF strip. It is important to note the orientation of the IEF strip so that the gel is face-up. We prefer to use sample cups to load protein samples. We do not recommend including the protein sample in the rehydration solution as this leads to vertical and horizontal streaks and protein loss (Zhou et al., 2005).

123

124

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Step 1 Casting second-dimension gel 1.1 Assemble gel cassette 1.2 Set up gradient maker with stopcocks closed and the outlet tube positioned between the gel plates 1.3 Position magnetic stirrer under front chamber (the one closest to the outlet) 1.4 Mix Heavy and Light acrylamide solutions according to Table I 1.5 Pour acrylamide solution into mixer chambers: Heavy solution in front chamber, Light solution in back chamber 1.6 Open chamber outlet and mixing channel stopcocks 1.7 Overlay gel with n-butanol 1.8 Allow the gel to polymerize for ≥ 1 h

FIGURE 4 Flowchart of Step 1.

Table 1  Composition of Acrylamide Solutions Needed to Form a 10–15% Gradient Gel Stock solution

Light solution

Heavy solution

Acrylamide (30% T, 2.6% C) 1.5 M Tris, pH 8.8 Sucrose SDS (20% w/v) H 2O APS (10% w/v) TEMED

8.25 mL 6.25 mL

12.25 mL 6.25 mL 3.75 g 125 μL 4.175 mL 82.5 μL 8.25 μL

125 μL 10.375 mL 82.5 μL 8.25 μL

5  Protocol

Tip

It is important to note the orientation of the IEF strip so that the gel is face-down during rehydration and face-up during isoelectric focusing. Note the serial number of each strip relative to each sample applied.

5.3  Step 3—Sample Preparation Overview

Duration 3.1A

3.1B

3.2

Tip Tip Tip

Tip

Sample preparation is the most crucial step of this protocol. 2D DIGE is a very sensitive method for detecting protein differences between samples. Care must be taken to limit the introduction of artifactual differences. This is particularly true for dissected tissues and subfractionated samples. Variability in preparation of these types of samples can lead to artifacts. Variable, depending on the sample. Minimal labeling of proteins: Place the cells or tissue sample in a 1.5-mL centrifuge tube that has a fitted pestle. Sufficient material to yield 100–200 μg protein is needed. Rinse the cells with an isotonic, ice-cold, low-salt Na-HEPES (pH 8.0) buffer that does not contain primary amines. Remove excess liquid and add 80 μL of cell lysis buffer with DDT. Homogenize the cells with a few passes or turns of the pestle. Centrifuge the sample in a microcentrifuge for 5–15 min at 15,000 × g at 4 °C to remove unbroken cells and debris. Saturation labeling of proteins: Place the cells or tissue sample in a 1.5-mL centrifuge tube that has a fitted pestle. Sufficient material to yield 1–3 μg protein is needed. Rinse the cells with an isotonic, ice-cold, low-salt Na-HEPES (pH 8.0) buffer. Remove excess liquid and add 10 μL of cell lysis buffer without DTT. Homogenize the cells with a few passes or turns of the pestle. Centrifuge the sample in a microcentrifuge for 5–15 min at 15,000 × g at 4 °C to remove unbroken cells and debris. Measurement of protein concentration using a Bradford assay. As urea, CHAPS and DTT affect the Bradford assay, the standards and samples should all be made up in lysis buffer. Add 1 μL sample/standard to 400 μL water in a plastic test tube and mix. Add 100 μL Bradford reagent, mix and measure the OD at 595 nm within 1 h. If the sample is too concentrated, dilute with lysis buffer. A standard curve should be made with BSA dissolved in lysis buffer at a concentration range of 0.1–2.5 mg/mL. The blank should be made with 1 μL lysis buffer. Keep the samples on ice throughout the lysis procedure. Homogenized samples can be stored at −80 °C for extended periods of time. Ideally the protein concentration of the lysate should be 1–2 mg/mL. If the lysate concentration is too high, dilute with lysis buffer. If the concentration is too low the sample may be concentrated by precipitation, but this could create problems with protein loss during precipitation, resolubilization and pH adjustment. It is preferable to start with sufficient material to yield a suitably concentrated lysate. Another important consideration is the removal of contaminating factors that affect IEF, such as salt, lipids, detergents and nucleic acids. There are a number of commercial protein cleanup kits that are worth testing; however, one must be cautious about protein loss and pH effects. We recommend labeling the protein samples first and then combining the samples before using a protein cleanup protocol.

See Fig. 5 for the flowchart of Step 3.

125

126

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Step 3 Sample preparation 3.1A Minimal labeling of samples containing 100-200 µg protein Lyse sample in cell lysis buffer with DTT

3.1B Saturation labeling of samples containing 1-3 µg of protein Lyse sample in cell lysis buffer without DTT

3.2 Measure protein concentration using Bradford assay

FIGURE 5 Flowchart of Step 3.

5.4  Step 4A—Minimal Protein Labeling with Lysine-Reactive Dyes Overview

Duration 4A.1 4A.2 4A.3 4A.4

4A.5

4A.6 4A.7 4A.8 4A.9

Tip Tip

The following four steps describe a typical DIGE experiment in which samples composed of 100–200 μg of protein are to be compared, denoted as sample “A” and “B.” In this case, the proteins are labeled with minimal labeling dyes, where Cy3-NHS and Cy5-NHS are used to label a minimal number of lysine residues. Set up four 1.5-ml microcentrifuge tubes, numbered 1–4. About 1 h. Pipette a volume of less than 75 μL of protein sample A, which contains 100–200 μg of protein in lysis buffer, into tubes 1 and 3. Pipette a volume of less than 75 μL of protein sample B, which contains 100–200 μg of protein in lysis buffer, into tubes 2 and 4. If needed, add a volume of cell lysis buffer with DDT to tubes 1–4 to bring the total volume up to 75 μL. Add 1 μL Cy3-NHS stock solution to tubes 1 and 4. Mix by vortexing. Briefly centrifuge to collect liquid at the bottom of the tube. Incubate in the dark on ice for 15 min. Add 1 μL Cy5-NHS stock solution to tubes 2 and 3. Mix by vortexing. Briefly centrifuge to collect liquid at the bottom of the tube. Incubate in the dark on ice for 15 min. To each tube, add 1 μL quenching solution. Mix by vortexing and briefly centrifuge. Incubate in the dark on ice for 30 min. In a new tube labeled “gel-1,” combine the entire contents of tubes 1 and 2. In a new tube labeled “gel-2,” combine the entire contents of tubes 3 and 4. Add 3.2 μL of the appropriate IPG buffer solution (for a 2% final IPG buffer concentration) to the gel-1 and gel-2 tubes. Mix by vortexing and briefly spin in a microcentrifuge at top speed for a few seconds at 4 °C to consolidate all of the liquid. Try to use equal amounts of protein in each sample. Optionally, one can add 1 μL of 1 mg/mL BSA in lysis buffer to each tube as a loading control. Mix by vortexing.

5  Protocol

Tip

Tip

Tip Tip

To synchronize the labeling time, add the dye to the walls of all reaction tubes. The Cy3 and Cy5 reaction tubes containing samples to be loaded on the same gel are briefly spun, vortexed and spun again in a microcentrifuge at top speed for a few seconds at 4 °C. To synchronize the quenching time, add the quencher to the walls of all reaction tubes. The Cy3 and Cy5 reaction tubes containing samples to be loaded on the same gel are briefly spun, vortexed and spun again in a microcentrifuge at top speed for a few seconds at 4 °C. The duration of quenching can be extended up to 2 h if necessary. The samples should be loaded onto IEF strips immediately after combining. After labeling, the samples can be stored without combining the Cy3- and Cy5-labeled samples at −80 °C. Thaw on ice before applying to the IEF strips.

See Fig. 6 for the flowchart of Step 4A.

5.5  Step 4B Saturation Protein Labeling with Cysteine-Reactive Dyes Overview

Duration 4B.1 4B.2 4B.3 4B.4 4B.5

4B.6

4B.7 4B.8 4B.9 4B.10

Tip

The following four steps describe a typical DIGE experiment in which samples composed of 1–3 μg of protein are to be compared, denoted as sample “A” and “B.” In this case, the proteins are labeled with saturation labeling dyes, where Cy3-Mal and Cy5-Mal are used to label a maximal number of cysteine residues. Set up four 1.5-mL microcentrifuge tubes, numbered 1–4. About 2 h. Pipette a volume of less than 75 μL of protein sample A that contains 1–3 μg of protein in lysis buffer into tubes 1 and 3. Pipette a volume of less than 75 μL of protein sample B that contains 1–3 μg of protein in lysis buffer into tubes 2 and 4. If needed add a volume of cell lysis buffer without DDT to tubes 1–4 to bring the total volume up to 75 μL. Add 1 μL of TCEP solution to tubes 1–4. Mix by vortexing. Briefly centrifuge to collect liquid at the bottom of the tube. Incubate in the dark at 37 °C for 60 min. Add 2 μL Cy3-Mal stock solution to tubes 1 and 4. Mix by vortexing. Briefly centrifuge to collect liquid at the bottom of the tube. Incubate in the dark at 37 °C for 30 min. Add 2 μL Cy5-Mal stock solution to tubes 2 and 3. Mix by vortexing. Briefly centrifuge to collect liquid at the bottom of the tube. Incubate in the dark at 37 °C for 30 min. To each tube, add 1 μL 7 M DTT solution. Mix by vortexing and briefly centrifuge. Incubate in the dark at 37 °C for 30 min. In a new tube labeled “gel-1,” combine the entire contents of tubes 1 and 2. In a new tube labeled “gel-2,” combine the entire contents of tubes 3 and 4. Add 3.2 μL of the appropriate IPG buffer solution (for a 2% final IPG buffer concentration) to the gel-1 and gel-2 tubes. Mix by vortexing and briefly spin in a microcentrifuge at top speed for a few seconds at 4 °C to consolidate all of the liquid. Try to use equal amounts of protein in each sample.

127

128

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Step 4A Minimal protein labeling with lysine-reactive dyes 4A.1 Aliquot 100-200 µg of sample A into tubes #1 and #3 4A.2 Aliquot 100-200 µg of sample B into tubes #2 and #3 4B.3 Add cell lysis buffer without DTT to bring volume of each tube to 75 µl

4A.4 Add 1 µl Cy3-NHS to tubes #1 and #4 Vortex and centrifuge Incubate in the dark for 15 min on ice

4A.5 Add 1 µl Cy5-NHS to tubes #2 and #3 Vortex and centrifuge Incubate in the dark for 15 min on ice 4A.6 Add 1 µl quenching solution to each tube Vortex and centrifuge Incubate in the dark for 30 min on ice

4A.7 Transfer the contents of tubes #1 and #2 to a new tube labeled ‘gel-1’

4A.8 Transfer the contents of tubes #3 and #4 to a new tube labeled ‘gel-2’ 4A.9 Add 3.2 µl IPG buffer matched to pH range of IEF strips Vortex and centrifuge

FIGURE 6 Flowchart of Step 4A.

5  Protocol

Tip Tip

Tip

Tip Tip

It is very important to make sure the cell lysis buffer used for saturation labeling does not contain DTT as this will inactivate the dyes. To synchronize the labeling time, add the dye to the walls of all reaction tubes. The Cy3 and Cy5 reaction tubes containing samples to be loaded on the same gel are briefly spun, vortexed and spun again in a microcentrifuge at top speed for a few seconds at 4 °C. To synchronize the quenching time, add the quencher to the walls of all reaction tubes. The Cy3 and Cy5 reaction tubes containing samples to be loaded on the same gel are briefly spun, vortexed and spun again in a microcentrifuge at top speed for a few seconds at 4 °C. The duration of quenching can be extended up to 2 h if necessary. The samples should be loaded onto IEF strips immediately after combining. After labeling, the samples can be stored without combining the Cy3- and Cy5-labeled samples at −80 °C. Thaw on ice before applying to the IEF strips.

See Fig. 7 for the flowchart of Step 4B.

5.6  Step 5—First-Dimension Electrophoresis: IEF Overview

Duration 5.1 5.2 5.3 5.4

5.5

5.6 5.7 Tip Tip

Tip

The isoelectric focusing step separates proteins according to their isoelectric point, pI. IEF should be set up according to the manufacturer’s instructions with a few modifications. Refer to the user manual for more details and precautions to be taken while setting up IEF. Less than 30 min contact time; 8–18 h electrophoresis time. Turn on the IEF unit. Place strip holder on the platform, making sure the electrode orientation is correct. Fill strip holder with the prescribed volume of strip covering fluid. Immerse the 3 MM paper wicks in HPLC water. The wicks should be approximately the width of the gel, typically 3 × 20 mm. Open the rehydration cassette assembly. Remove a rehydrated IEF strip and rinse it briefly with HPLC water. Carefully blot excess water. Place the IEF strip gel side up in the holder with the acidic end toward the anode. Blot excess water from the wicks. Place wicks at both ends of the strip. Place an electrode on each of the wicks. Place a sample cup near the electrode on the acidic end of the gel. Pipette the sample into the sample cup, taking care to avoid bubbles. Place the lid over the strip holder. Start the IEF program according to the manufacturer’s instructions. We typically use the following conditions for pH 3–10 NL,18-cm strips (see Table 2). The IEF machine is covered with an opaque cover to limit photobleaching. The time taken to reach 30–40 kVh using the parameters listed in Table 2 is between 8 and 18 h. The duration depends on the sample and can vary from run to run depending on the amount of salt and other contaminants in the sample. Wash all IEF accessories on finishing IEF. Use double-distilled water for washing. Wash strip holders with the IEF strip-cleaning detergent, using cotton swabs to void scratching. Rinse thoroughly with water and then absolute ethanol. Wash electrodes with water and ethanol. Clean strip-holder lids and the IPGphor platform with ethanol to remove any covering fluid.

129

130

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Step 4B Saturation protein labeling with cysteine-reactive dyes 4B.1 Aliquot 1-3 µg of sample A into tubes #1 and #3 4B.2 Aliquot 1-3 µg of sample B into tubes #2 and #3 4B.3 Add cell lysis buffer without DTT to bring volume of each tube to 75 µl 4B.4 Add 1 µl TCEP solution to each tube Vortex and centrifuge Incubate in the dark at 37 C for 60 min 4B.5 Add 2 µl Cy3-Mal to tubes #1 and #4 Vortex and centrifuge Incubate in the dark at 37 C for 30 min 4B.6 Add 2 µl Cy5-Mal to tubes #2 and #3 Vortex and centrifuge Incubate in the dark at 37 C for 30 min

4B.7 Add 1 µl 7M DTT solution to each tube Vortex and centrifuge Incubate in the dark at 37 C for 30 min 4B.8 Transfer the contents of tubes #1 and #2 to a new tube labeled ‘gel-1’

4B.9 Transfer the contents of tubes #3 and #4 to a new tube labeled ‘gel-2’ 4B.10 Add 3.2 µl IPG buffer matched to pH range of IEF strips Vortex and centrifuge

FIGURE 7 Flowchart of Step 4B.

5  Protocol

See Fig. 8 for the flowchart of Step 5. Table 2  Recommended Program for Running 18-cm, pH 3–10 IEF Strips Step 1 2 3 4 Total

Voltage 500 1000 8000 8000

Voltage step

Duration (h)

Duration (kVh)

Step-n-hold Gradient Gradient Step-n-hold

1–8 1–4 3 2–3

0.5–4 0.75–3 13.5 16–24 30.75–44.5

Step 5 First-dimension electrophoresis IEF 5.1 Turn on IEF unit Position strip holder on platform 5.2 Fill strip holder with prescribed volume of strip covering fluid 5.3 Soak paper wicks in water 5.4 Remove IEF from rehydration tray and rinse in water Blot away excess water Position strip in strip holder gel-side up 5.5 Blot excess water from wicks Position wicks, electrodes and sample cup on IEF strip 5.6 Pipette the sample into the sample cups Place lid on the strip holder 5.7 Start electrophoresis program (Table II) FIGURE 8 Flowchart of Step 5.

131

132

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

5.7  Step 6—Equilibration Overview

Duration 6.1

6.2

6.3 6.4 6.5 6.6 Tip Tip

The transition from the first- to second-dimension electrophoresis steps requires the exchange of high-concentration urea and thiourea for SDS and lower concentration urea. At the same time, the proteins are reduced with DTT and alkylated with iodoacetamide. 1 h. One hour before the completion of the IEF step, prepare equilibration solutions. For each IEF strip dissolve 100 mg of DTT in 10 mL of equilibration buffer (equilibration buffer + DTT). For each IEF strip dissolve 250 mg of iodoacetamide (IAA) in 10 mL of equilibration buffer (equilibration + IAA buffer). Immediately at the completion of the IEF program, remove the electrodes and wicks from the IEF strips. Using fine forceps, transfer each IEF strip to an equilibration dish by curling the strip along the inside of the dish with the gel surfaces facing inward. Add 10 mL of room temperature equilibration + DTT buffer. Place the dish on a rotating mixer and gently swirl for 15 min. Drain completely. Rinse briefly with distilled water. Drain completely. Add 10 mL of room temperature equilibration + IAA buffer. Place the dish on a rotating mixer and gently swirl for 15 min. Drain completely. Equilibrated IEF strips should either be run immediately on the second dimension or sealed in the equilibration dish and stored at −80 °C. The IEF strip gel material is very delicate. Be extra cautious to avoid nicking or tearing the gel. Another option for an equilibration chamber is to use long tubes that accommodate the IEF strips. Equilibration tubes are commercially available.

See Fig. 9 for the flowchart of Step 6.

5.8  Step 7—Second-Dimension Electrophoresis Overview

Duration 7.1

7.2 7.3

The second-dimension electrophoresis is done in the presence of SDS, which separates proteins based on their size. The most important consideration of this step is to quickly transfer the equilibrated IEF strip to the second-dimension gel ensuring good contact between the gel of the IEF strip and the second-dimension gel. About 30 min contact time; 6–18 h electrophoresis time. Once the first equilibration step is under way, melt agarose-sealing solution in a microwave and place in a beaker of hot water to keep melted. Place the molten agarose in a 65 °C water bath or heating block to keep the agarose in a liquid state. Drain the n-butanol from the top of the second-dimension gel and rinse well with water. Drain completely. Remove the IEF strip from the IAA + equilibration solution or thaw if previously equilibrated and frozen. Place IEF strip on top of the seconddimension gel with the plastic touching the back glass of the gel cassette. Orient the acidic end of the strip toward the left. Trim the ends of the strip if needed. Gently push the IEF strip down until it contacts the stacking gel.

5  Protocol

Step 6 Equilibration 6.1 Prepare equilibration solutions; for each strip: Add 100 mg DTT to 10 ml equilibration buffer (equilibration buffer + DTT) Add 250 mg IAA to 10 ml equilibration buffer (equilibration buffer +IAA) 6.2 Remove sample cups, electrodes and wicks from completed IEF strips Position strip in equilibration dish with gel facing inward 6.3 Add 10 ml equilibration buffer + DTT Swirl for 15 min and drain completely 6.4 Briefly rinse with water and drain completely 6.5 Add 10 ml equilibration buffer + IAA Swirl for 15 min and drain completely 6.6 Immediately run IEF strip on second-dimension gel or store at -80 C

FIGURE 9 Flowchart of Step 6.

7.4 7.5

7.6

7.7

Caution Tip

Cover the IEF strip with approximately 1 mL of melted agarose until it just covers the IEF strip, avoiding formation of air bubbles. Place the gel cassette in the electrophoresis unit and fill the upper and lower chambers with running buffer. The lower tank should be filled with 3.5 L running buffer and stirred constantly at 4 °C in a cold room. Electrophorese at a constant current with a maximum voltage set at 500. Typically the current is set at 10–25 mA per gel depending on how long one wants the run to take (e.g. 25 mA per gel requires approximately 10 h to complete and 15 mA per gel takes approximately 16 h). At the completion of electrophoresis, remove the gel from the glass plates. The dye front and IEF strip should be removed, and the gel soaked in fixative for ≥2 h with gently swirling. Be sure to vent the sealing agarose tube while in the microwave. Avoid boiling over this solution. It is important to carry out this transfer step as quickly as possible to minimize protein diffusion in the IEF strip.

133

134

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Tip Tip Tip

Tip Tip

Gel cassette with a taller back-plate is easier to load than cassettes with equal height plates. The IEF strip gel material is very fragile. Be extra cautious to avoid nicking or tearing the gel. Make sure the gel side is not touching the front glass. Dispense the agarose from each end so that it covers evenly. Make sure there are no bubbles. If there are bubbles, remove them once the agarose solidifies. Work quickly to prevent the agarose from solidifying unevenly. Gels can be stored in fixative for several months without significant loss of fluorescent signal. For long-term storage, refrigeration is recommended. However, protein spots to be identified by MS should be cut out and frozen at −80 °C within 1 week.

See Fig. 10 for the flowchart of Step 7. Step 7 Second-dimension electrophoresis

7.1 Melt agarose sealing solution in microwave Place in 65 C heating block

7.2 Drain n-butanol from second-dimension gel Rinse with water and completely drain 7.3 Place equilibrated IEF strip on top of second-dimension gel 7.4 Cover IEF strip with ~1 ml of sealing agarose solution 7.5 Place gel cassette in electrophoresis unit Fill tank with tank buffer 7.6 Electrophorese at constant current until dye front runs off the gel 7.7 Transfer the completed gel to fixative solution

FIGURE 10 Flowchart of Step 7.

5  Protocol

5.9  Step 8—Image Acquisition Overview Duration 8.1

Tip Tip

Tip

Tip

To visualize the fluorescently tagged proteins in the 2DE gels, the gels must be scanned with a fluorescent gel-imaging system. About 40 min per gel. Acquire images using your chosen imaging system. The DIGE experiment described will yield two or four images: either one two-color image from gel-1 and reciprocal gel-2 or two single-color images each from gel-1 and gel-2. Each image is typically 1024 × 1280 pixels, with a resolution of 135 μm per pixel. These images should be stored as raw unsigned 16-bit data as to maximize portability for image analysis. There are two main imager options: commercial or homemade. We use a home-built device that has an integrated spot picking robot. Commercial systems utilize separate devices for gel imaging and spot picking. There are two main reasons for poor results: insufficient protein labeling and incomplete IEF. Insufficient labeling is commonly due to the pH of the lysate being <8.0 or the presence of excess primary amines, such as Tris. If there is sufficient sample, one can test the pH using a small piece of pH paper. Tris <50 mM does not appear to interfere with protein labeling and 30 mM is recommended. Incomplete focusing is usually the result of interfering contaminants, such as salts and nucleic acids. Salts, which can be removed using a protein cleanup kit, prevent the IEF from reaching the desired voltage. Nucleic acids make the sample viscous and difficult to handle and also interfere with focusing. Nucleic acids can be removed by commercially available cleanup kits or can be broken up by sonication or the addition of nuclease. There will always be gel-to-gel variation for 2DE gels. This is due to a myriad of uncontrollable features in manufacturing, sample preparation and electrophoretic conditions. DIGE was developed to circumvent some of this variability. The digital-imaging system and fluorescence labeling also provide a three or four orders of magnitude linear response, which provides much more accurate estimation of protein abundance than silver or Coomassie blue staining of proteins. DIGE protein differences can be visualized in a variety of ways, including color overlay (Fig. 1) and two-frame looping movies (see Step 9).

See Fig. 11 for the schematic representation of the home-built gel imager.

5.10  Step 9—Image Analysis Overview

Duration

Once the separate Cy3 and Cy5 images are acquired, one must analyze the images to detect protein changes. These changes can be at the level of protein abundance or posttranslational changes. About 1 h per gel.

135

136

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

FIGURE 11 Fluorescence gel imager/spot picker. This diagram illustrates the components of the DIGE gel imager. This device comprises the following components: a scientific-grade Peltiercooled 16-bit CCD camera (Photometrics/Roper Scientific); an 105-mm macrolens (Nikon); a computer-driven stage (New England Affiliated Technology); and a spot-picker drive (Applied Precision). All electronic components are controlled by a computer workstation running Windows and custom software. Not shown is the illumination system, which is composed of two 250-W quartz-tungsten-halogen light sources (Oriel) that direct their light through motorized filter wheels (Ludl). The light is directed onto the gel via fiber-optic light guides (Oriel), and fluorescence wavelengths are selected by band-pass filters (Chroma Technology). See the color plate. 9.1

9.2

To perform image analysis in our laboratory, we employ several software applications: ImageJ, QuickTime and SExtractor. ImageJ is used to convert the raw 16-bit images into contrast-adjusted JPEG images. These images are either viewed as a two-frame animation in ImageJ or ported to QuickTime. QuickTime allows one to modulate the frame rate of the animation. To quantify protein differences, subregions of the gel images containing protein differences are analyzed by SExtractor, a free astronomical imageanalysis package. This program automatically detects and quantifies protein spots. The outcome of image analysis is a list of difference-protein spots that indicate significant differences between the two protein samples being compared.

5  Protocol

Tip

Tip Tip

Tip

In general, we rely on the two-frame looping movies to identify the significant protein changes we want to identify by MS. The exogenously added BSA serves as a loading control to balance the image display parameters for making two-frame looping movies. More precise quantification is done later using SExtractor. To accurately assess the level of protein change, the Cy3 and Cy5 images are summed and submitted to SExtractor for spot detection, which generates a list of elliptical objects that specify individual protein spots. This list is used as a mask to determine the protein spot intensities in background-subtracted Cy3 and Cy5 images. This approach typically yields an SD of 5–7% for unchanging spots. Visual difference-protein detection requires <1 h for an experienced user to complete. We prefer viewing animated two-frame movies over color overlays or three-dimensional spot rendering to detect protein differences. SExtractor parameters must be adjusted to properly detect protein spots, which is somewhat different from the default settings for stars and galaxies. Commercial spot-detection packages are also available. These are quite reliable, but require some manual editing. The commercial packages are particularly useful when comparing multiple gels.

5.11  Step 10—Spot Picking Overview

Duration 10.1 10.2

10.3 Tip Tip

Tip

Once protein differences are detected, the protein spot is excised from the 2DE gel and prepared for MS identification. The following description is specific for our home-built imager. For commercial devices, follow the manufacturer’s instruction. About 1 h. Fill the wells of a 96-well receiving plate with 1% acetic acid solution. Use the integrated spot-cutting tool of the home-built imager to “click” on spots of interest; cut a 1.8-mm diameter plug from the gel and deposit it in a 96-well plate designed for a protein digestion robot. The imager software allows one to create a list of protein spots to be retrieved. After all the desired spots are cut, the acetic acid solution is removed and the plate moved to the digestion robot. The wells should be brimming with solution to ensure proper ejection of the gel plug. A key difference between the gel-imaging system we use and commercial imaging systems is that we use an open-gel format that is amenable to imaging and spot picking within the same device, whereas commercial imagers (such as GE’s Typhoon Imager) are best suited to imaging the gel within the electrophoresis plates, so spot picking is done on a different device. The open-gel format does not require the gel to be adhered to one of the electrophoresis plates. The cut gel fragments can be stored at −80 °C for at least several weeks.

137

138

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

5.12  Step 11—Protein Identification Overview Duration 11.1

Tip

Tip

Tip

Tip

Tip

The final step of the protocol is to identify the isolated proteins by MS. This step is dependent of the type of MS used for identification. Depends on the number of spots and the type of MS used. Identify the difference proteins using MS. This can be done by routine methods using a variety of instruments. Consult your preferred MS facility director. We generally use MALDI-TOF. Minimal DIGE labeling with Cy3-NHS or Cy5-NHS labels only a small fraction of proteins in the lysate: approximately 5% of all proteins have a single dye molecule bound, whereas the rest have none. Hence, minimal DIGE labeling does not affect the MS identification of proteins. Saturation DIGE labeling labels nearly all cysteine residues, thus MS identification of cysteine-containing peptides must be corrected for the mass of Cy3-Mal and Cy5-Mal. It is important to note, however, that the DIGE imaging system is capable of detecting <0.2 fmol of protein. To identify low-abundance difference proteins, one must either enrich for the proteins of interest or pool protein from multiple DIGE gels. MS/MS machines are also suitable. LC separation of peptides is not necessary since the peptide mixtures arising from single gel spot have very low complexity. Care should taken to avoid carryover of peptides between samples being analyzed. Blank controls should be run between samples.

Keywords Keyword class

Keyword

Methods List the methods used to carry out this protocol (i.e. for each step)

1. Two-dimensional electrophoresis 2. Difference gel electrophoresis 3. DIGE 4. Fluorescent labeling 5. Cy dyes 6. Minimal labeling 7. Saturation labeling 8. Isoelectric focusing 9. SDS PAGE 10. Fluorescence imaging 11. Image analysis 12. Spot cutting 13. Mass spectrometry 1 2 3 4 5

Process List the biological process(es) addressed in this protocol

Rank

Snippet 1 2 3 4 5 6 7 8 9 10 11 12 13

5  Protocol

Keyword class

Keyword

1 Organisms List the primary organism used in 2 this protocol. List any other 3 applicable organisms 4 5 1 Pathways List any signaling, regulatory, or 2 metabolic pathways addressed 3 in this protocol 4 5 1 Molecule roles List any cellular or molecular 2 roles addressed in this protocol 3 4 5 1 Molecule functions List any cellular or molecular 2 functions or activities addressed 3 in this protocol 4 5 1 Phenotype List any developmental or 2 functional phenotypes addressed 3 in this protocol (organismal or 4 cellular level) 5 1 Anatomy List any gross anatomical 2 structures, cellular structures, 3 organelles, or macromolecular 4 complexes pertinent to this 5 protocol Diseases List any diseases or disease processes addressed in this protocol Other List any other miscellaneous keywords that describe this protocol

1 2 3 4 5 1 2 3 4 5

Rank

Snippet

139

140

CHAPTER 6  Two-Dimensional Difference Gel Electrophoresis (2D DIGE)

Acknowledgments The development of DIGE would not have been possible without the efforts and dedication of my students and staff: Mustafa Ünlü, Melissa Krajcovic, Liz Morgan, Chris Lacenere, Surya Viswanathan, Lei Gong, Mamta Puri, Anupam Goyal, and Susan Dowd.

References Source article(s) used to create this protocol Referenced literature Gong, L., Puri, M., Unlu, M., Young, M., Robertson, K., Viswanathan, S., et al. (2004). Drosophila ventral furrow morphogenesis: a proteomic analysis. Development, 131, 643–656. Gorg, A., Postel, W., & Gunther, S. (1988). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 9, 531–546. Hamdan, M., & Righetti, P. G. (2005). Proteomics today: Protein assessment and biomarkers using mass spectrometry, 2D electrophoresis, and microarray technology. Hoboken, N.J. John Wiley & Sons. Klose, J. (1975). Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik, 26, 231–243. Kondo, T., Seike, M., Mori, Y., Fujii, K., Yamada, T., & Hirohashi, S. (2003). Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool. Proteomics, 3, 1758–1766. Lilley, K. (2002). Protein profiling using two-dimensional difference gel electrophoresis (2-D DIGE). In J. E. Coligan, B. M. Dunn, D. W. Speicher, & P. T. Wingfield (Eds.), Current protocols in protein science. (pp. 22.22.1–22.22.14). New York: John Wiley & Sons, Inc. Lilley, K. S., & Friedman, D. B. (2004). All about DIGE: quantification technology for differential-display 2D-gel proteomics. Expert Review of Proteomics, 1, 401–409. Marouga, R., David, S., & Hawkins, E. (2005). The development of the DIGE system: 2D fluorescence difference gel analysis technology. Analytical and Bioanalytical Chemistry, 382, 669–678. O’Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry, 250, 4007–4021. Okano, T., Kondo, T., Kakisaka, T., Fujii, K., Yamada, M., Kato, H., et  al. (2006). Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis. Proteomics, 6, 3938–3948. Scheele, G. A. (1975). Two-dimensional gel analysis of soluble proteins. Characterization of guinea pig exocrine pancreatic proteins. Journal of Biological Chemistry, 250, 5375–5385. Shaw, M. M., & Riederer, B. M. (2003). Sample preparation for two-dimensional gel electrophoresis. Proteomics, 3, 1408–1417. Sitek, B., Potthoff, S., Schulenborg, T., Stegbauer, J., Vinke, T., Rump, L. C., et al. (2006). Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling. Proteomics, 6, 4506–4513. Ünlü, M., Morgan, M. E., & Minden, J. S. (1997). Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis, 18, 2071–2077.

5  Protocol

Viswanathan, S., Ünlü, M., & Minden, J. S. (2006). Two-dimensional difference gel electrophoresis. Nature Protocols, 1, 1351–1358. Wang, H., Clouthier, S. G., Galchev, V., Misek, D. E., Duffner, U., Min, C. K., et al. (2005). Intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids. Molecular and Cellular Proteomics, 4, 618–625. Zhou, G., Li, H., DeCamp, D., Chen, S., Shu, H., Gong, Y., et al. (2002). 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Molecular and Cellular Proteomics, 1, 117–124. Zhou, S., Bailey, M. J., Dunn, M. J., Preedy, V. R., & Emery, P. W. (2005). A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure. Proteomics, 5, 2739–2747.

Related literature Cramer, R., & Westermeier, R. (2012). Difference gel electrophoresis (DIGE): methods and protocols. New York: Humana Press; Springer. Sheehan, D., & Tyther, R. (2009). Two dimensional electrophoresis protocols. New York, NY London: Humana Press; Springer.

141