In Vitro Transcription and Translation Coupled to Two-Dimensional Electrophoresis for Bacterial Proteome Analysis

Summary

The most popular approach for proteomic analysis is based on the combination of two-dimensional electrophoresis (2DE) and mass spectrometry. Although very effective, the method suffers from a number of limitations, the most serious one being the necessity of expensive and sophisticated instrumentation to be operated by skilled personnel. Here, we propose an alternative approach, which is particularly useful when one is interested to establish if a subset of proteins is present in a complex protein mixture derived from a sequenced organism. The method is based on amplification of the genes whose products are under investigation. The amplified genes are used in transcription and translation reactions and the derived radio-labeled proteins are separated by 2DE. The gel is autoradiographed and the autoradiograph is superimposed on the 2D gel (sample gel) from which the protein mixture from the organism has been separated. The matching between the autoradiographic spots and the protein spots of the sample gel allows immediate protein identification.

1 Introduction

Global analysis of proteins from cells and tissues is termed “proteomics.” Although a variety of alternative procedures have been developed, at present one of the most popular approaches for proteomic analysis requires the previous knowledge of the genome sequence of the organism under investigation and is based on the combination of two-dimensional gel electrophoresis (2DE) and mass spectrometry. According to this approach, proteins are separated by 2DE, stained, in-gel digested with trypsin or other proteolytic enzymes and finally subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The MALDI-TOF analysis provides peptide mass fingerprints, which lead to protein identification when compared with the theoretical in silico fingerprints generated from the available genome sequence. Usually, 80–85% of the analyzed protein spots give a mass fingerprint, which is in most cases sufficient for protein identification. A limited number of spots (approx 5%) requires further tandem mass spectrometry analysis (MS/MS) for unambiguous characterization.

The major drawback of this approach is that it requires expensive and sophisticated instruments, which need to be operated by well-trained and specialized scientists. In addition, the method presents some limitations in sensitivity, not owing to mass spectrometers (which can analyze samples in the low fmole range), but to sample preparation procedures, which are usually inefficient, making the analysis feasible only when protein quantities greater than 0.1–0.2 pmole are available (1). We have recently described an alternative method for the characterization of proteomes, in particular bacterial proteomes, which may offer some advantages over the current proteomic approaches (2). The method combines PCR cloning, in vitro transcription-translation and 2DE. In this chapter, we describe two applications of the method, one aimed at identifying a single protein in the two-dimensional (2D) maps, the second designed to simultaneously identify a set of proteins.

1.1 Single Protein Identification in 2D Maps

When the scope of the investigation is to establish whether a specific protein is present in a complex protein mixture, for instance in the total protein extract of a given bacterium, the following procedure is proposed ( Fig. 1 ). The bacterium is grown under appropriate conditions and the bacterial culture is used to (1) prepare the mixture of cellular proteins ( Fig. 1 , step 1a) and (2) purify chromosomal DNA ( Fig. 1 , step 1b). Chromosomal DNA is used to amplify the gene encoding the protein under investigation ( Fig. 1 , step 2) and the amplified gene is used to drive the in vitro expression of the radio-labeled protein ( Fig. 1 , step 3). The transcription and translation reaction (TTR) is analyzed by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) for a qualitative and quantitative evaluation ( Fig. 1 , step 4). TTR is then mixed with the total cellular proteins and the mixture is separated by 2DE ( Fig. 1 , step 5). The 2D gel is then stained with Coomassie Blue ( Fig. 1 , step 5). Because a relatively small amount of TTR is added to the bacterial total protein sample (the proper amount of TTR is estimated by analytical monodimensional electrophoresis and autoradiography, and corresponds to the amount sufficient to visualize the labeled translated protein after overnight exposure) the proteins of the Escherichia coli S-30 cell extracts do not show up on the gel and the only visible spots derive from the bacterial protein mixture under examination. After staining, the 2D gel is autoradiographed ( Fig. 1 , step 6) and the autoradiograph is finally superimposed on the stained gel ( Fig. 1 , step 7). The protein spot, which eventually matches the spot visible on the autoragiograph, corresponds to the protein encoded by the amplified gene used in the TTR.

Fig. 1.

Schematic representation of the procedure for the identification a single specific protein 2D map. The protein mixture under investigation (protein sample, step 1a) and chromosomal DNA (step 1b) are prepared starting from the same bacterial culture. Chromosomal DNA is used for the amplification of the gene whose product is under investigation and the amplified gene is either cloned in an appropriate expression vector or utilized as linear fragment for in vitro protein synthesis (step 2). TTR is carried out in the presence of ¹⁴C-labeled leucine and lysine (step 3). A small aliquot of the reaction is analyzed by SDS-PAGE for a qualitative and quantitative evaluation (step 4). The protein sample obtained from the bacterial culture is then mixed with a small aliquot of TTR, and the mixure is resolved by two-dimensional electrophoresis (step 5). The 2D-gel is stained with Colloidal Coomassie Blue, and then dried and autoradiographed (step 6). Finally, autoradiograph and gel are superimposed (step 7). The gel spot that matches with the spot on the autoradiography corresponds to the protein encoded by the amplified gene used in the TTR.

1.2 Multiple Protein Identification in 2D Maps

The procedure described for single protein identification is not amenable for the simultaneous identification of several protein spots. In fact, this would require the addition of several TTRs to the protein mixture under analysis. In so doing, the amounts of S-30 E. coli proteins would be high enough to be visualized after Coomassie Blue staining, thus making the subsequent identification of the protein spots in the sample mixture very complicated.

To overcome this problem, the following procedure has been developed ( Fig. 2 ). A bacterial culture is used for protein sample ( Fig. 2 , step 1a) and chromosomal DNA preparation ( Fig. 2 , step 1b). Chromosomal DNA is used for gene amplification and the amplified genes are either cloned in appropriate expression vectors or utilized as linear fragments for in vitro protein synthesis ( Fig. 2 , step 2). In vitro TTRs are carried out in the presence of ¹⁴C-labeled amino acids ( Fig. 2 , step 3). Small aliquots of each reaction are analyzed by SDS-PAGE for a qualitative and quantitative evaluation, and TTRs are properly pooled on the basis of the SDS-PAGE analysis ( Fig. 2 , step 4). Pooled TTRs and the protein sample are separately resolved by 2DE in the presence of the same protein markers, and the 2D gels are Coomassie Blue-stained ( Fig. 2 , step 5). The 2DE gel of the TTRs is then dried and autoradiographed for TTR visualization, and the position of the protein markers are reported on the autoradiograph ( Fig. 2 , step 6). Finally, the TTR autoradiograph and sample gel are superimposed by computer-assisted image analysis using the protein markers as landmarks ( Fig. 2 , step 7). Spot matching between radioactive spots on the autoradiograph and Coomassie-stained protein spots on the protein sample allow the identification of protein spots on the sample gel (see Note 1 ).

Fig. 2.

Schematic representation of the strategy for multiple protein identification on a 2D map. The protein mixture under investigation (protein sample, step 1a) and chromosomal DNA (step 1b) are prepared starting from the same bacterial culture. Chromosomal DNA is used for the amplification of the genes of interest and the amplified genes are added to in vitro transcription and translation reactions for the synthesis of the corresponding proteins (step 2). TTRs are carried out in the presence of ¹⁴C-labeled leucine and lysine (step 3). Small aliquots of each reaction are analyzed by SDS-PAGE for a qualitative and quantitative evaluation, and TTRs are properly pooled on the basis of the SDS-PAGE analysis (step 4). Pooled TTRs and protein sample are separately resolved by 2DE in the presence of protein markers, and the 2D gels are stained with Coomassie Blue (step 5, protein marker spots represented by open circles [yellow]). The 2D gel on which the mixture of the transcription and translation reactions (TTRs) has been resolved is dried and autoradiographed for the visualization of the radioactive in vitro synthesized proteins (gray circles [red], step 6). The position of the protein markers (open circles [yellow]) are reported on the autoradiograph (step 6). Finally, the TTR autoradiograph and sample gel are superimposed by computer-assisted image analysis using the protein markers as landmarks (step 7). The protein spots that match with the radioactive spots correspond to the products of the genes used for the TTRs.

In the following sections, we will first provide detailed protocols for multiprotein identification in 2D maps and then the methods for single protein identification.

2 Materials

2.1 Device, Growth Medium, and Buffers for Neisseria meningitidis Outer Membrane Protein

• 1. Device for bacteria lysis. Bacterial cells are disrupted with a French Press apparatus (SLM Instruments, Inc., Rochester, NY).

• 2. Bacteria growth medium. Bacteria are grown on GC agar plates (BD Biosciences, Franklin Lakes, NJ) supplemented with 4 g/L glucose, 0.1 g/L glutamine, and 2.2 mg/L cocarboxylase at 37°C in a humidified atmosphere containing 5% CO₂.

• 3. Bacteria lysis and wash buffers. Bacterial cells were washed in 40 mM Tris-base and cell lysis was carried out in 40 mM Tris-base containing 1000 U of Benzonase (Sigma, St. Louis, MO).

2.2 PCR Amplification and Cloning of the Genes of Interest

PCR amplification is performed with Pwo DNA polymerase (Roche Diagnostics GmbH, Mannheim, Germany). Genes of interest were cloned into the plasmid pET-21b+ (Novagen, Madison, WI). Plasmid preparations were carried out using the Qiagen kit (Qiagen GmbH, Hilden, Germany).

2.3 2DE Devices

First-dimension isoelectric focusing is performed using devices from Amersham Biosciences (Uppsala, Sweden). The devices include an Immobiline Dry-Strip Reswelling Tray, IPGphor strip holders, and an IPGphor Isoelectric Focusing System. Second-dimension Gradient polyacrylamide SDS-PAGE electrophoresis is performed using devices from Bio-Rad (Hercules, CA). The devices include a Mini-Protean II Multi-Casting Chamber, Model 485 Gradient Former and a Mini-Protean II Cell.

2.4 Mixture for In Vitro Transcription/Translation Reactions

• 1. ¹⁴C-labeled amino acids. L-[U-¹⁴C] leucine and L-[U-¹⁴C] lysine at 11.7 and 12.2 Gbq/mmol, respectively, are purchased from Amersham Biosciences.

• 2. Amino acid mixture. The mixture includes each of the 20 amino acids, with the exception of leucine and lysine, at a concentration of 1 mM in diethylpyrocarbonate (DEPC)-treated water. The amino acid mixture is stable for more than 1 yr when stored at −20°C. DEPC-treated water is prepared by adding 1 mL of DEPC per liter of bidistilled water; the solution is stirred for 1 h and then autoclaved.

• 3. Low molecular weight (LMW) mixture. A 470-µL stock of LMW mixture can be prepared and stored in 100-µL aliquots at −20°C. It will be stable for several months, and can be thawed and frozen a few times. The composition of the LMW is prepared from stock solutions as described in Table 1 . All stock solutions are kept at −20°C and are stable for several months. The stock solutions labeled with an asterisk are autoclaved.

• 4. Reaction mixture. The reaction mixture is prepared before use as described in Table 2 .

Table 1 LMW Mixture

Table 2 Reaction Mixture

2.5 Mixtures for 2DE

• 1. Acrylamide mixture. The acrylamide mixtures are prepared before use; the amounts given are necessary for the preparation of 12 gels (1.5 mm thick, 7.3 cm high, 8.3 cm wide). Fifty milliliters of 0.8% (w/v) piperazine di-acrylamide (PDA) are prepared freshly in 30% (w/v) acrylamide. The mixtures are prepared from stock solutions as indicated in Table 3 .

• 2. Reswelling solution. The solution is prepared freshly prior to use. One hundred twenty five microliters per sample are needed. The composition is: 7 M urea, 2 M thiourea, 2% (w/v) 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate hydrate (CHAPS), 2% (w/v) 3-[N,N-dimethyl (3-myristoylaminopropyl)ammonio]propanesulfonate amidosulfobetaine-14 (ASB-14), 2% (v/v) IPG nonlinear pH 3.0–10.0 buffer, 2 mM tributyl-phosphine (from 1 M stock solution in isopropanol), 65 mM DTT.

• 3. Reequilibration solution. The solution is prepared freshly prior to use. Twelve milliliters per sample are needed. The composition is 50 mM Tris-HCl, pH 8.8, 4 M urea, 2 M thiourea, 2.5% (w/v) acrylamide, 30% (w/v) glycerol, 2% (w/v) SDS, 5 mM tributyl-phosphine, traces of Bromo phenol Blue (3).

• 4. Running buffer: 198 mM glycine, 125 mM Tris, 0.1% (w/v) SDS. The buffer is prepared from 10X stock solution. Dilution is prepared prior to use.

• 5. Agarose solution: 0.5% (w/v) agarose in running buffer containing 2 mM tributyl-phosphine. The solution is prepared freshly prior to use.

• 6. Colloidal Coomassie staining, solution I: 2% (v/v) H₃PO₄, 50% (v/v) methanol (4). The solution is prepared freshly prior to use.

• 7. Colloidal Coomassie staining, solution II: 2% (v/v) H₃PO₄, 34% (v/v) methanol, 17% (w/v) (NH₄)₂SO₄ (4). The solution is prepared freshly prior to use.

• 8. Water-saturated butanol. Combine in a bottle 50 mL of butanol with at least 5 mL of water. Use top phase to overlay gels. Store at room temperature indefinitely.

Table 3 Acrylamide Solutions

2.6 Devices and Solutions for Autoradiography

• 1. Devices. Gels are dried on 3MM Chr paper (Whatman International Ltd., Kent, England) using a Bio-Rad gel dryer Model 583. Autoradiography cassettes and BioMax MR-2 film are from Eastman Kodak Co., Rochester, NY.

• 2. Autoradiography enhancer solution: 125 mM salicyclic acid in 40% methanol.

• 3. Autoradiograph developer and fixer: 1X GBX developer and 1X GBX fixer are from Eastern Kodac Co.

2.7 Software for Image Acquisition and Analysis

The software used for image acquisition and analysis is provided by Amersham Biosciences. Images of autoradiographs are acquired with the Personal Densitometer SI. Radioactive signal relative to each radio-labeled protein is quantified using ImageQuant 5.1. Image Master Elite software v3.10 was used for in silico superimposition of gel and autoradiography.

3 Methods

3.1 Multiprotein Identification in 2D Maps

As already described in the introduction, Fig. 2 summarizes the steps required for the simultaneous identification of several proteins on the same 2D map. The detailed protocol of each step is reported next.

3.1.1 Preparation of Bacterial Proteins (Step 1a) and Chromosomal DNA (Step 1b)

The protein sample used to illustrate the methodology is an outer membrane protein preparation of Neisseria meningitidis strain MC58 (2). Briefly, bacteria were grown to confluence on supplemented GC agar plates at 37°C in a humidified atmosphere containing 5% CO₂. Bacteria (approx 10¹¹ cells) were harvested from 10 plates and resuspended in 10 mL of 40 mM Tris. Chromosomal DNA was prepared according to standard procedures (5) from 1 mL of cell suspension and stored at a concentration of 1 mg/mL at −20°C. The remaining 9 mL were used for membrane protein preparation. Bacteria were inactivated by 45-min incubation at 65°C, cooled on ice in the presence of benzonase (1000 U), and bacterial cells were disrupted with the French Press apparatus at 18,000 psi. The lysate was centrifuged at 70,000g overnight at 5°C. The pellet was washed twice with 40 mM Tris-base and resuspended in 2 mL of reswelling solution. After centrifugation at 100,000g at 10°C for 3 h, the supernatant containing the solubilized outer membrane proteins was aliquoted and stored at −80°C. Typically, 10 mg of outer membrane proteins were obtained.

3.1.2 PCR Amplification and Cloning of the Genes of Interest

TTRs are usually carried out using circular plasmids as templates. Briefly, the genes of interest (see Note 1 ) were PCR amplified from Neisseria meningitidis strain MC58 chromosomal DNA using Pwo DNA polymerase and appropriate synthetic forward and reverse primers carrying the NdeI and XhoI (or HindIII) restriction sites for the insertion of the gene into the plasmid pET-21b+ (see Subheading 2.2. ). In pET-21b+ a stop codon was introduced upstream from the nucleotide sequence coding for the hexa-histidine tag to avoid the addition of the His-Tag to the C-terminus of the proteins of interest (see Note 2 ). Plasmid preparations were carried out using the Qiagen kit (see Subheading 2.2. ), and plasmid DNAs were stored at a concentration of 1 mg/mL at −20°C (see Note 3 ).

3.1.3 In Vitro TTRs

In vitro-coupled transcription-translations are carried out as described by Pratt (6), with minor modifications. Reactions are performed in 10 µL final volume containing 20–25 µg/mL plasmid DNA or 8–16 µg/mL linear DNA (see Subheading 3.1.2. ), 0.42 mM ¹⁴C-labeled L-leucine and L-lysine, and 4 mg/mL of E. coli S30-extract proteins (see Note 4 ).

• 1. For 10 TTRs, add in a microfuge tube 42 nmol of radioactive leucine and lysine.

• 2. Reduce the volume of the radioactive amino acids to 1–2 µL with a SpeedVac apparatus (Savant, Holbrook, NY).

• 3. Add 80 µL of reaction mixture into the microfuge tube containing the radioactive amino acids.

• 4. Mix and split the reaction mixture into 10 TTR tubes (8 µL per tube).

• 5. Add to each tube the DNA of interest (20–25 µg/mL plasmid or 8–16 µg/mL linear fragment, volume should be about 2 µL).

• 6. Allow the reaction to proceed for 3 h at 37°C (see Note 5 ).

• 7. Add 90 µL of cold ethanol to each tube and incubate for 30 min at −20°C.

• 8. Mix and transfer 15 µL of each reaction into new tubes for SDS-PAGE analysis, while the remaining 75 µL are used for 2DE.

• 9. Recover the proteins by 10 min centrifugation at 13,000g.

• 10. Dry protein pellets with a SpeedVac apparatus (Savant), and solubilize them for SDS-PAGE or 2DE (see Subheadings 3.1.4. , steps 1 and 12, respectively).

3.1.4 Quantification and Mixing of TTRs

• 1. Dissolve the pellets from the 15-µL samples (see Subheading 3.1.3. , steps 8–10) with 5 µL of SDS-PAGE loading buffer.

• 2. Heat the samples for 3 min at 100°C.

• 3. Resolve proteins by standard SDS-PAGE using a 12.5% polyacrylamide gel.

• 4. Soak the gel with the autoradiography enhancer solution (see Subheading 2.6. ).

• 5. Dry the gel on 3MM Chr paper using the gel dryer (see Subheading 2.6. ).

• 6. Autoradiograph the gel overnight using BioMax MR-2 film, at −80°C, in the autoradiography cassettes.

• 7. Develop the film 2–3 min with autoradiograph developer (see Subheading 2.6. ).

• 8. Wash the autoradiograph with water.

• 9. Fix the autoradiograph 1 min with autoradiograph fixer (see Subheading 2.6. ).

• 10. Acquire the image of the film with a Personal Densitometer SI. To guarantee a sufficiently high resolution, we routinely acquire the image at 50 µm per pixel and 12 bits.

• 11. Quantify the radioactive signal relative to each radio-labeled protein using ImageQuant 5.1 software.

• 12. Based on quantification, normalize the radioactive signals and mix appropriate aliquots of each TTR (see Note 6 ).

3.1.5 2DE of Protein Sample and TTRs

Protein sample and TTRs (see Note 1 ) are mixed with reference proteins (see Note 7 ). Sample proteins and TTRs are then separated in the first dimension on nonlinear pH 3.0–10.0 (7 cm) IPG strips (see Note 8 ), and in the second dimension on 9–16.5% linear gradient polyacrylamide SDS gels (1.5 mm thick, 7.3 cm high, 8.3 cm wide). Gels are stained with Colloidal Coomassie Blue.

• 1. Cast two gels using the Mini-Protean II Multiple-Casting Chamber and develop the acrylamide gradient by gravity using the gradient Former.

• 2. Cover the gels with butanol saturated with water, cover the Mini-Protean II Multiple-Casting Chamber with Parafilm (American National Can™, Menasha, WI) and allow the gels to polymerize overnight.

• 3. Mix the protein sample (see Subheading 3.1.1. ) with 1 µg of each protein marker and bring the mixture to a final volume of 125 µL with reswelling solution (see Subheading 2.5. ).

• 4. Mix TTR pool (see Subheading 3.1.4. , step 12 and Note 6 ) with 1 µg of each reference protein and bring the mixture to a final volume of 125 µL with reswelling solution.

• 5. Before gel loading, dissociate possible protein aggregates by subjecting the mixture to three consecutive cycles of 5-min sonication in a sonicator bath and vigorous agitation.

• 6. Transfer the protein solutions (protein sample and TTR mixture) into two track lanes of the Immobiline Dry-Strip Reswelling Tray.

• 7. Cover each solution with an IPG strip (gel side down), without trapping air bubbles under the strips.

• 8. Overlay the strips with 1.5–3 mL of IPG cover fluid (Amersham Biosciences).

• 9. Allow strip hydration to proceed overnight.

• 10. Transfer the IPG strips to the IPGphor strip holders.

• 11. Place filter paper pads at the end of the gel strips.

• 12. Cover the IPGphor strip holders with IPG cover fluid.

• 13. Place the IPGphor strip holders on the IPGphor Isoelectric Focusing System, and focalize the proteins absorbed in the strips by applying the following voltage: 150 V for 35 min, 500 V for 35 min, 1000 V for 30 min, 2600 V for 10 min, 3500 V for 15 min, 4200 V for 15 min, and then 5000 V to reach 10 kVh.

• 14. After isoelectric-focusing, transfer each strip into 15-mL Falcon tubes and equilibrate each strip for 10 min in 6 mL of reequilibration solution.

• 15. Repeat step 14 .

• 16. Remove the butanol saturated with water from the top of the polymerized gels (one for the sample protein and the other for the TTR mixture [see step 1 ]).

• 17. Wash the surface of the gels with water.

• 18. Cover the gels with 0.5 mL agarose solution.

• 19. Lay the strips on top of the agarose bed (one per gel).

• 20. Cover the strips with the agarose solution and allow solidification at room temperature.

• 21. Place the gels in the Mini-Protean II Cell and run the second dimension in the running buffer by applying 35 mA per gel until the blue fronts reach the end of the gels.

• 22. Wash the gels for 10 min in H₂O.

• 23. Fix the gels for 60 min in the Colloidal Coomassie staining, solution I.

• 24. Wash the gels three times for 10 min in H₂O.

• 25. Incubate the gels for 1 h in the Colloidal Coomassie staining, solution II.

• 26. Add 0.065 g of Coomassie G250 per 100 mL of Colloidal Coomassie staining, solution II.

• 27. Allow the staining to develop for 48 h.

• 28. Destain the gels with water until the background staining is removed (usually, three consecutive washes of 10 min each are sufficient).

• 29. Acquire the image of the 2D gel on which the sample proteins have been resolved using a high-resolution densitometer (see Subheading 3.1.4. , step 10).

• 30. Soak the gel with the autoradiography enhancer solution.

• 31. Dry the TTR gel as described in Subheading 3.1.4. , step 5.

3.1.6 Autoradiography of 2DE Gel

• 1. Fix the dried TTR gel on the bottom of the autoradiography cassette using adhesive paper.

• 2. In the dark, lay a BioMax MR-2 film on top of the gel and fix it using adhesive paper.

• 3. Carefully and precisely mark the position of the film with respect to the gel. This step is critical because, after development and fixing, the autoradiograph has to be relocated on the gel exactly in the same position as the one used during autoradiography.

• 4. Expose the film overnight at −80°C.

• 5. Develop and fix the film as described in Subheading 3.1.4. , steps 7–9.

• 6. Lay the developed, dried film back on the gel in its original position using the markers to properly align the film with the gel.

• 7. Using black ink, precisely mark the positions of the protein markers on the autoradiograph.

• 8. Acquire the digital image of the autoradiograph with marked protein markers as described in Subheading 3.1.4. , step 10.

3.1.7 In Silico Superimposition of TTR Film and Sample 2D Gel

For an accurate matching process, a computer program should be used that allows matching the digitalized images using protein markers as landmarks. The rationale is to ask the computer algorithm to correct all possible distortions occurred during protein separations by 2D-electrophoresis by forcing the protein markers to coincide. The result of this process is the artificial creation of a single combined gel in which the autoradiographic spots are positioned in the 2D gel of the protein sample where they would have migrated if the TTRs would have been run on the same gel together with the protein sample. The protein spots of the sample gel, which eventually coincide with, or are in close proximity of, the autoradiographic spots correspond to the proteins encoded by the genes used for the TTRs.

For in silico superimposition of gel and autoradiography, we used the software Image Master Elite v3.10 (see Subheading 2.7. and Note 9 ). An example of matching is given in Note 10 . For those familiar with this software the steps are as follows:

• 1. Create a combined gel in which the TTR autoradiograph is superimposed on the sample gel using protein markers as landmarks.

• 2. Scan the combined gel for protein sample spots coinciding with or located in close proximity to TTR spots, by using increasing vector box size (see Note 10 ).

• 3. The protein spots that are closest to the autoradiographic spots (namely, that require the smallest vector box size to give a match [see Note 10 ]), are the proteins with the highest likelihood to be encoded by the genes used for the TTR reactions.

3.2 Single Protein Identification in 2D Maps

Figure 2 summarizes the steps required for the identification of a single specific protein on a 2D map. The detailed protocol for each step is reported next:

• 1. Bacterial proteins (step 1a) and chromosomal DNA preparations (step 1b). Follow the procedure described in Subheading 3.1.1.

• 2. PCR amplification and cloning of the gene of interest. Follow step 2 described in Subheading 3.1.2.

• 3. In vitro TTR. Follow the procedure described in Subheading 3.1.3. with the following modifications:

  • a. Add 4.2 nmol of radioactive leucine and lysine to a microfuge tube.

  • b. Dry the radioactive amino acids in a SpeedVac apparatus (Savant).

  • c. Add 8 µL of reaction mixture to the microfuge tube containing the dried radioactive amino acids and mix.

  • d. Add 2 µL of DNA (200–250 ng if circular plasmid is used, or 80–160 ng if linear, amplified fragment is used).

  • e. Allow the reaction to proceed for 3 h at 37°C (see Note 5 ).

  • f. Add 90 µL of cold ethanol and keep at −20°C for 30 min.

  • g. Mix and transfer 15 µL into a new tube to be subsequently used for SDS-PAGE analysis. The remaining 75 µL will be used for 2DE.

  • h. Precipitate the proteins in both tubes by 10 min centrifugation at 13,000g.

  • i. Dry protein pellets with a Speed Vac, and solubilize them for SDS-PAGE or 2DE (see Subheadings 3.1.4. , steps 1 and 12, respectively).

3.3 Analysis of the TTR by SDS-PAGE

The analysis of the TTR by SDS-PAGE is recommended to define the correct amount of TTR to be subsequently added to the protein sample (see Subheading 3.4. , step 3) to have a visible autoradiographic spot after overnight exposure of the 2D gel.

• 1. Dissolve the pellet from the 15-µL sample (see Subheading 3.2. step 3, item g) with 5 µL of SDS-PAGE loading buffer.

• 2. Heat the sample for 3 min at 100°C.

• 3. Resolve proteins by standard SDS-PAGE using a 12.5% polyacrylamide gel.

• 4. Soak the gel with the autoradiography enhancer solution (see Subheading 2.6. ).

• 5. Dry the gel on 3MM Chr paper using the gel dryer.

• 6. Autoradiograph the gel overnight using BioMax MR-2 film, at −80°C, in an autoradiography holder.

• 7. Develop the film 2–3 min with autoradiograph developer.

• 8. Wash the autoradiograph with water.

• 9. Fix the autoradiograph 1 min with autoradiograph GBX fixer.

• 10. Acquire the image of the film with a densitometer such as the Personal Densitometer SI. To guarantee a sufficiently high resolution, we routinely acquire the image at 50 µm per pixel and 12 bits.

• 11. Quantify the radioactive signal relative to each radio-labeled protein using ImageQuant 5.1. Add to the protein sample (see Subheading 3.4. , step 3) a sufficient amount of TTR to allow the visualization of only the major translation product of the reaction after overnight autoradiographic exposure of the 2D gel. Usually, this is obtained by adding between 5 and 20 µg of TTR (see Note 11 and Subheading 3.4. ).

3.4 2DE of Protein Sample and TTR Mixture

• 1. The day before the electrophoretic separation, cast one gel using the Mini-Protean II Multi-Casting Chamber and develop the acrylamide gradient by gravity using the gradient Former.

• 2. Cover the gel with butanol saturated with water (see Subheading 2.5. ), cover the Mini-Protean II Multiple-Casting Chamber with Parafilm and allow the gels to polymerize overnight.

• 3. The day after gel casting, add up to 20 µg of TTR (see Subheading 3.2. , step 3i and Note 12 ) to 200 µg of protein sample (see Subheading 3.2.1. ) and bring to a final volume of 125 µL with reswelling solution.

• 4. Before gel loading, dissociate possible protein aggregates by subjecting the mixture to three consecutive cycles of 5-min sonication in a sonicator bath and vigorous agitation.

• 5. Transfer the protein solution into a track lane of the Immobiline Dry-Strip Reswelling Tray.

• 6. Cover the solution with an IPG strip (gel side down), without trapping air bubbles under the strip.

• 7. Overlay the strip with 1.5–3 mL of IPG cover fluid (Amersham Biosciences).

• 8. Allow the strip hydration to proceed overnight.

• 9. Transfer the IPG strip to the IPGphor strip holder.

• 10. Place filter paper pads at the end of the gel strip.

• 11. Cover the IPGphor strip holder with IPG cover fluid.

• 12. Place the IPGphor strip holder on the IPGphor Isoelectric Focusing System, and focalize the proteins absorbed in the strip by applying the following voltage: 150 V for 35 min, 500 V for 35 min, 1000 V for 30 min, 2600 V for 10 min, 3500 V for 15 min, 4200 V for 15 min, and then 5000 V to reach 10 kVh.

• 13. After isoelectric-focusing, transfer the strip into a 15-mL Falcon tube and equilibrate the strip in 6 mL of reequilibration solution (see Subheading 2.5. ) for 10 min.

• 14. Repeat step 13 .

• 15. Remove the butanol saturated with water from the top of the polymerized gel (see step 1 ).

• 16. Wash the surface of the gel with water.

• 17. Cover the gel with 0.5 mL agarose solution.

• 18. Lay the strip on top of the agarose bed.

• 19. Cover the strip with the agarose solution and allow solidification at room temperature.

• 20. Place the gels in the Mini-Protean II Cell and run the second dimension in the running buffer by applying 35 mA until the blue front reaches the end of the gels.

• 21. Wash the gel for 10 min in H₂O.

• 22. Fix the gel for 60 min in the Colloidal Coomassie staining, solution I.

• 23. Wash the gel three times for 10 min in H₂O.

• 24. Incubate the gel for 1 h in the Colloidal Coomassie staining, solution II.

• 25. Add 0.065 g of Coomassie G250 per 100 mL of Colloidal Coomassie staining, solution II.

• 26. Allow the staining to develop for 48 h.

• 27. Destain the gel with water until the background staining is removed (usually, three consecutive washes of 10 min each are sufficient).

• 28. Soak the gel with the autoradiography enhancer solution.

• 29. Dry the gel as described in Subheading 3.1.4. , step 5.

3.5 Autoradiography of the 2DE Gel

• 1. Fix the dried TTR gel on the bottom of the autoradiography cassette (Eastman Kodak Co.), using adhesive paper. In the dark, lay a BioMax MR-2 film (Eastman Kodak Co.) on top of the gel and fix it using adhesive paper.

• 2. Carefully and precisely mark the position of the film with respect to the gel. This step is critical because, after development and fixing, the autoradiograph has to be relocated on the gel exactly in the same position as the one used during autoradiography.

• 3. Expose the film overnight at −80°C.

• 4. Develop and fix the film as described on Subheading 3.1.4. , steps 7–9.

• 5. Lay the developed, dried film back on the gel in its original position using the markers to properly align the film with the gel. The protein spot(s) matching the radioactive spot(s) correspond(s) to the product of the gene used for TTR.

4 Notes

• 1. In the example given in Fig. 3 , we performed TTRs from six genes selected from the genome of Neisseria meningitidis serogroup B (MenB) (2), for which we were interested to know whether the corresponding proteins were present in a MenB outer-membrane preparation. The selected genes encode the proteins NMB1710 (Glutamate dehydrogenase, NADP-specific), NMB1972 (Chaperonin, 60kDa), NMB1936 (ATP synthetase F1, α subunit), NMB1429 (outer membrane protein PorA), NMB2039 (outer membrane protein PIB), and NMB0382 (outer membrane protein class 4).

• 2. In the case of proteins with leader sequence, the genes were amplified by replacing the leader sequence portion with the methionine start codon. The presence of leader sequences was established by using Psort, available at http://psort.nibb.ac.jp/form.html.

  For example, the two genes NMB1972 and NMB0382 identified in the 2D map of Neisseria meningitis (see Note 10 and Fig. 4 ) were amplified using the following primers:

  • FOR-NMB197

  • 5′-AGAATTCCATATGGCAGCAAAAGACG TACAGTTCGGCA-3′,

  • REV-NMB1972

  • 5′-ATACCGCTCGAGTCACATCATGCCGCCCAT ACCACCCA-3′,

  • FOR-NMB0382

  • 5′-GGAATTCCATATGGGCGAGGCGTCCGTTCAG GGTTACAC-3′,

  • and REV-NMB0382

  • 5′-ATACCGCTCGAGTTAGTGTTGGTGATGAT TGTGTGCCGG-3′

  (The NdeI and XhoI sites used for the insertion of the PCR fragments into the plasmid pET-21b+ (Novagen, Madison, WI) are underlined). Cloning was performed according to standard procedures (5). Plasmid preparations were carried out using the Qiagen kit (Qiagen GmbH, Hilden, Germany), and plasmid DNAs were stored at a concentration of 1 mg/mL at −20°C.

• 3. TTRs can also be performed using linear DNA fragments as template (2). It has to be pointed out that if DNA linear fragments are used, the addition of extra nucleotides at the extremities of the fragments are required. This is to prevent the T7 promoter and terminator from being degraded by nucleases present in the S30-extract during TTR. For instance, when we included the 78 nucleotides and 103 nucleotides that in pET21b+ precede and follow the T7 promoter and terminator, respectively, we did not observe a substantial impairment of the transcription activities of the fragments during the 3-h incubation.

• 4. Different S30-extracts are commercially available (e.g., Promega, Madison, WI; Qiagen, Hilden, Germany) and are well suited for the procedure. We prepared the S30-extract in house, using the E. coli strain BL21DE3 (Invitrogen, Carlsbad, CA.) containing endogenous T7 RNA polymerase. The S30-extract preparation was performed as described by Pratt (6), adapting the protocol to 2 L of culture. Usually, 8 mL of S30-extract at a protein concentration of 12–15 mg/mL is obtained. When stored at −80°C, this amount of S30-extract is stable for 3–4 mo and is sufficient for more than 2400 TTRs.

• 5. To enhance protein synthesis, 8 µL of reaction mixture, where the S30-extract is replaced by H₂O, can be added to each TTR and the reaction continued for additional 3 h.

• 6. It is important that each in vitro translated radioactive protein shows up with similar intensities on the 2D map. To achieve that, normalization by SDS-PAGE analysis is an important step. For instance, Fig. 3 shows the autoradiograph after 14 h exposure of an SDS-gel on which 15 µL of 6 TTRs are resolved as described in Subheading 3.1.4. The scanning analysis performed on the autoradiograph indicated that the relative amounts of the major products of each reaction, normalized with respect to lane 6, were: 1.3, 2.0, 1.5, 2.1, and 0.7 (lanes 1, 2, 3, 4, and 5, respectively). Therefore, if the six TTRs were to be analyzed on the same 2D gel, to visualize only the predominant product of each reaction at the same intensity, the following volumes of each reaction should be mixed: 11.5, 7.5, 10.0, 7.1, 21.4, and 15.0 µL. As an example, we show in Fig. 4B the autoradiograph of a 2D gel on which 7.5 and 15 µL of the TTR products of lanes 2 and 6 in Fig. 3 were separated after mixing. The major reaction products, which resolved in more than one spot as frequently happens in 2DE, showed up at similar intensities (see boxed spots), and no intermediate TTR products were visible.

• 7. Addition of protein markers to both protein samples and TTRs is a fundamental step of the procedure in that the protein markers must be subsequently utilized to properly superimpose the sample gel to the TTR gel. Protein markers should be selected to have an even distribution throughout the gel. This in fact allows the software algorithm to compensate the local gel distortions with higher accuracy. Although commercial kits for 2DE protein markers are available, we made our own set of protein markers consisting in highly purified recombinant proteins with molecular weights and pIs values ranging from 21.8 to 84.0 kDa, and from 4.89 to 8.62, respectively. The list of the selected proteins is given in Table 4 .

• 8. Different IPG strips having broad or narrow pI ranges are commercially available. We recommend the use of broad pI range strips because theoretical and experimental pI values do not always coincide. If a better resolution is needed, narrow-range pI strips can be used once the pI value has been experimentally determined.

• 9. Analysis with Melanie III has provided comparable satisfactory results.

• 10. To illustrate the effectiveness of the multi-protein identification procedure, the identification of two meningococcal proteins is reported in Fig. 4 . In this particular experiment, we were interested in establishing whether the products of the NMB1972 and NMB0382 genes were present in an outer-membrane protein preparation of Group B N. meningitidis. Figure 4A shows the Coomassie Blue-stained 2D gel on which the membrane proteins were separated together with the mixture of protein markers (1 µg each, labeled in green). Figure 4B shows the autoradiograph of a 2D gel (TTR gel), run in parallel with the gel of Fig. 4A , in which the two TTRs reactions, carried out using the NMB1972 and NMB0382 genes as templates, were separated in the presence of the same protein markers as the ones used in the gel of Fig. 4A . In Fig. 4B , the autoradiographic spots corresponding to the radioactive NMB1972 and NMB0382 proteins are boxed in red (see Note 6 ), whereas the blue spots correspond to the protein markers whose positions were marked upon superimposition of the autoradiograph on the TTR gel stained with Coomassie Blue. The images of the gel in Fig. 4A and of the autoradiograph in Fig. 4B were scanned, digitalized and subsequently in silico-superimposed using the protein markers as landmarks. Figure 4C represents the combined image derived from the in silico superimposition of Fig. 4A,B . In essence, the computer superimposes the autoradiograph on the protein sample gel using markers as reference spots and in so doing compensates the distortions that occurred during gel running. The result of this process is that the radioactive spots change their position in the virtual combined image according to the corrections. Once proper compensations are done, the computer “scans” around the radioactive spots in search for the nearest protein spots belonging to the protein sample gel and indicates the distance (vector box size) between the center of the radioactive spots and the nearest protein spot. For instance, in Fig. 3C the matching between the autoradiographic spots of NMB1972 and NMB0382 (red spots) and the Comassie Blue stained spots (green circles) of the MenB membrane protein gel are zoomed. As shown, the centers of the red and green circles are located only 12 and 13 vector box sizes apart, respectively. Because one box size corresponds to 50 µm in length, practically speaking, the superimposed spots almost coincide. The conclusion of this experiment was that both NMB1972 and NMB0382 were present in the membrane protein preparation; on the 2D map, the NMB1972 was resolved in six isoforms whereas NMB0382 was constituted by two isoforms.

• 11. It is very important to load a limited amount of TTR on the gel so that the proteins of the E. coli S30-extract do not show up on the gel. This is why the preliminary analysis of TTR using SDS-PAGE and autoradiography is required: it allows to establish the minimum amount of TTR sufficient to visualize the radioactive spots after overnight exposure. In our experience, anything below 20 µg of TTR does not give visible spots under the conditions we used for the Coomassie Blue staining.

• 12. It has to be considered that in a complex protein mixture, completely unrelated proteins having similar molecular weights and isoelectric points could be present. Because these proteins tend to comigrate on 2D gels, the possibility that the products of the TTRs find a match with an unrelated protein cannot be ruled out. However, on the basis of our experience in proteomic analysis using 2DE coupled to MALDI-TOF, we have estimated that only 1–2% of all visible spots are constituted by more than one protein. Therefore, statistically, only 1 out of 100 protein identifications is expected to be incorrect.

Fig. 3.

SDS-PAGE analysis of the products of 6 TTRs. The six Neisseria meningitidis genes NMB1710 (lane 1), NMB1972 (lane 2), NMB1936 (lane 3), NMB1429 (lane 4), NMB2039 (lane 5), NMB0382 (lane 6) were amplified and used for in vitro synthesis of the encoded proteins. Six micrograms of each TTR were separated by SDS-PAGE. The gel was then dried and autoradiographed (see Note 6 for details).

Table 4 List of Protein Markers

Fig. 4.

Identification of NMB1972 and NMB0382 proteins in the 2D map of Neisseria meningitidis outer membrane proteins. (A) Coomassie-Blue staining of the 2D gel of the total N. meningitidis outer membrane proteins run together with 22 protein markers (see Table 4 ). The protein markers (1µg each) are labeled in green. (B) Autoradiograph of the TTRs used for in vitro labeling of NMB1972 and NMB0382 proteins. The two TTRs were separated on a two dimensional (2D) gel together with the same protein markers as in A and the gel was autoradiographed. On the autoradiograph the position of the protein markers are labeled in blue. (C) Computer-derived image obtained by in silico superimposition of the 2D gel image shown in A on the TTR autoradiograph shown in B. The zoomed boxes indicate the matching of the autoradiographic spots with the gel spots. (For details see Note 10 .)