Identification of differentially expressed proteins in human malignant pleural effusions

Patients

The present study was approved by the Institutional Review Board of Mackay Memorial Hospital (Taipei, Taiwan) and written informed consent was obtained from all participants. A total of 27 patients with pleural effusion were included, of whom 14 had a cytologically positive malignant pleural effusion and 13 a transudative effusion. The malignant effusions were due to lung cancer in nine patients (eight adenocarcinoma and one small cell carcinoma), metastatic tongue cancer in two, metastatic oesophageal cancer in one and metastatic breast cancer in two. The transudates were attributed to congestive heart failure in 10 patients, nephrotic syndrome in one and liver cirrhosis in two.

Pleural fluid (∼150–200 mL) was collected from each of the participants by echo-guided aspiration. The effusion was centrifuged for 10 min at 800×g at 4°C, and the supernatant stored in aliquots at -70°C until further analysis.

Protein preparation

Total protein was precipitated from each sample using a 2-D Clean-UP Kit (Amersham Biosciences, Uppsala, Sweden). Briefly, 100 μL pleural effusion was mixed with 300 μL precipitant and incubated on ice for 15 min. Then, 300 μL co-precipitant was added to the mixture, which was centrifuged for 10 min at 8,000×g. The pellet was resuspended in 500 μL co-precipitant and centrifuged for 5 min at 8,000×g. The final product was obtained from the pellet remaining after centrifugation for a further 10 min at 8,000×g.

Precipitated proteins were prepared for Sypro Ruby (Molecular Probes, Eugene, OR, USA) staining or fluorescence (CyDye; Amersham Biosciences) labelling before proceeding to two-dimensional gel electrophoresis (2-DE). For Sypro Ruby staining, 1 mg precipitated protein was resuspended in DeStreak Rehydration Solution (Amersham Biosciences) to 350 μL prior to isoelectric focusing (IEF). For CyDye labelling, precipitated proteins were resuspended in dissolving solution (4% 3-((3-cholamidopropyl)diethylammonio)-1-propane sulphonate (CHAPS), 30 mM tris(hydroxymethyl)aminomethane (Tris)–HCl (pH 8.5) and 7 M urea), and labelled with fluorescent cyanine dyes developed for fluorescence difference gel electrophoresis (Amersham Biosciences) according to the manufacturer’s protocol. Briefly, protein (50 μg) was labelled with 1 μL amine-reactive cyanine dye (400 pmol·µL^-1) freshly dissolved in anhydrous dimethyl formamide. The labelling reaction was incubated in the dark for 30 min at 4°C. The reaction was terminated by addition of 10 nmol lysine. Immobiline DryStrip rehydration buffer (7 M urea, 2 M thiourea, 2 mg·mL^-1 dithiothreitol and 1% pharmalytes) was added to make up the volume to 350 μL prior to IEF.

Two-dimensional gel electrophoresis

In order to identify proteins expressed differentially in malignant and transudative pleural effusions, the protein components of each sample were separated into a two-dimensional array using 2-DE. This was performed using IEF in the first direction (18-cm pH 3–10 and pH 4–7 Immobiline DryStrip (IPGphor; Amersham Biosciences)) and sodium dodecylsulphate (SDS)-polyacrylamide gel electophoresis (SDS-PAGE) in the second direction (Ettan DALTsix Large Vertical System; Amersham Biosciences). IEF was performed following a step-and-hold stepwise incremental voltage program: 30 V for 18 h, 500 V for 30 min, 1,000 V for 30 min, 1,500 V for 30 min, 2,000 V for 30 min, 3,000 V for 30 min, 6,000 V for 1 h, and 8,000 V for 10–13 h, giving a total power of 80,000 Vh. After IEF, the strips were subjected to two-step equilibration in buffers containing 6 M urea, 2% weight (w)/volume (v) SDS, 30% w/v glycerol and 50 mM Tris–HCl (pH 8.8), with 1% w/v dithiothreitol for the first step and 2.5% w/v iodoacetamide for the second step. The strips were then transferred on to SDS-PAGE gels (1.0 mm thick; 12% T, where T is the combined percentage (w/v) of acrylamide and bisacrylamide) and electrophoresed for 4 h at 17 W·gel^-1 at 15°C. For samples with Sypro Ruby staining, the gel was washed in 10% methanol/7% acetic acid for ∼1 h and placed in Sypro Ruby for ≥3 h. Samples studied by difference gel electrophoresis were labelled with CyDyes before proceeding to 2-DE.

Image acquisition and analysis of two-dimensional electrophoresis gels

The pattern of protein expression in each 2-DE gel was studied using an image analyser following image acquisition by a scanner (Typhoon^TM 9400 Imager; Amersham Biosciences). The density of each protein spot on the scanned image represented the level of protein expression. Quantification of spot density was performed using a specific image analyser.

For Sypro Ruby images, the gels were scanned at an excitation wavelength of 610–630 nm in order to visualise all protein spots present in the gel. Prior to analysis, images were cropped to remove areas extraneous to the gel image using ImageQuant^TM V5.2 (Amersham Biosciences). The density of each differentially expressed spot was quantified by PDQuest Image Analysis software V7.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

For CyDye-labelled images, the Cy3 images (green fluorescence; malignant effusion) were scanned using a 532-nm laser and a 580-nm band-pass (BP) 30 emission filter, the Cy5 images (red fluorescence; transudative effusion) were scanned using a 633-nm laser and a 670-nm BP30 emission filter, and the Cy2 images (blue fluorescence; internal control) were scanned using a 488-nm laser and a 520-nm BP40 emission filter. The resolution was set to 100 μm for all gels. After acquisition, images were merged and analysed using DeCyder^TM V5.0 (Amersham Biosciences) differential analysis software. In the merged image, yellow fluorescence (green plus red) indicated a protein present at similar levels in malignant and transudative effusions, light green fluorescence indicated a protein present at higher levels in malignant effusions and orange fluorescence indicated a protein present at higher levels in transudative effusions. The fluorescent intensity of each spot was digitalised and converted into a three-dimensional image, allowing both quantitative and visual comparison of different spots.

In-gel tryptic digestion

In order to identify proteins of interest, the protein spots showing differential expression were removed and digested into peptides by proteases using the following method. The protein spots on 2-DE were excised from the gel manually and cut into pieces. Each gel piece was dehydrated independently in acetonitrile for 10 min, vacuum dried, rehydrated in 55 mM dithioerythreitol in 25 mM ammonium bicarbonate (pH 8.5) for 1 h at 37°C, and then alkylated with 100 mM iodoacetamide in 25 mM ammonium bicarbonate (pH 8.5) for 1 h at room temperature. The pieces were then washed twice, for 15 min each time, in 50% acetonitrile in 25 mM ammonium bicarbonate (pH 8.5), dehydrated in acetonitrile for 5 min, dried, and rehydrated in a total of 100 ng sequencing-grade modified trypsin (Promega, Madison, WI, USA) in 25 mM ammonium bicarbonate (pH 8.5) for 16 h at 37°C. Following digestion, each sample of tryptic peptides was extracted in 50% acetonitrile containing 5% formic acid for 15 min with moderate sonication. For optimal extraction, the procedure was repeated once more, then the extracted solutions were pooled and evaporated to dryness under vacuum. For MALDI-MS analysis, dry peptide samples were redissolved in 0.1% trifluoroacetic acid and purified on a C18 Zip-Tip (Millipore, Billerica, MA, USA) according to the manufacturer’s instruction manual.

Protein mass fingerprinting analysis of protein spots and sequence database search

The final protein recognition and confirmation were performed by matching the peptides identified by MALDI-Q-TOF MS. The sequences of eluted peptides were first identified by subjecting the samples to MALDI-Q-TOF analysis 5. Briefly, tryptic peptides from 2-DE protein spots were subjected to protein mass fingerprinting using a MALDI-Q-TOF mass spectrometer (M@LDI^TM; Micromass, Manchester, UK) operated in positive ion reflectron mode. Samples were spotted into a 96-well MALDI target plate using a saturated matrix solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/1% trifluoroacetic acid. Mass spectra were acquired for the mass range 800–3,000 Da and automatically processed by the Mascot search engine for protein mass fingerprinting against the Swiss-Prot database. The ion masses were submitted to the NCBInr database using the Mascot ions search for mass matching and verification of protein identity. The identification of a protein based on MS/MS analysis of one peptide was considered sufficient if the Mascot score was above the significance level. Positive identification of proteins required at least five matching peptide masses with ≥50 parts per million mass accuracy.

Direct high-precision analysis for protein identification and sequence database search

If any of the proteins identified by MALDI-Q-TOF MS appeared to show potential for further study, they were subjected to reconfirmation using a higher-precision method employing nano-liquid chromatography (LC) combined with nano-electrospray ionisation tandem MS (LC-MS/MS), as adapted from a previous study 5. Briefly, LC-MS/MS analyses were performed using an integrated LC-MS/MS system (Micromass). After data acquisition, the individual MS/MS spectra acquired for each of the precursors within a single LC run were output as a single Mascot-searchable peak list (.pkl) file. The peak list files were used to query the Swiss-Prot database using the Mascot program. Only significant hits, as defined by Mascot probability analysis, were initially considered.

Western blot analysis and ELISA

The expression of any selected protein was further studied in each pleural effusion by Western blotting and ELISA. Each protein sample was mixed with an electrophoresis buffer containing 2% SDS and 5% β-mercaptoethanol and boiled for 10 min. In order to ensure equal loading of each sample, protein quantification was performed using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc.). Briefly, 800 μL of each sample was added to 200 μL Bio-Rad protein assay dye reagent concentrate. All assays were performed in duplicate. After incubation for ≥5 min at room temperature, samples were quantified by measuring absorbance at 595 nm. Proteins (5 μg) were then separated on a 10% SDS-PAGE gel. The fractionated proteins were electroblotted on to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). The membranes were blocked using 5% bovine serum albumin in 0.1% Tween 20 in Tris-buffered saline (TBST) for ≥2 h and then incubated with a 1:1,000 dilution of primary antibody for 1 h. After washing in TBST, the membranes were incubated with peroxidase-conjugated secondary antibody for 1 h, and proteins were detected using an enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA, USA). All protein bands were analysed densitometrically using Multi Gauge Ver. 2.2 software (Fujifilm, Tokyo, Japan).

Measurement of pigment epithelium-derived factor levels in pleural effusions

Pigment epithelium-derived factor (PEDF) levels were studied in all samples by Western blot analysis and ELISA. Briefly, PEDF concentrations were determined using a Chemikine^TM PEDF sandwich ELISA kit (Chemicon International, Chandlers Ford, UK). In order to measure total PEDF, samples were treated with urea (8 M) and diluted 1:500 in assay diluents. PEDF levels were quantified by enzyme immunoassays according to the manufacturer’s protocol (Fuji Chemical Industries, Toyama, Japan). As there was substantial variation in protein quantities and fluid volumes among the different forms of pleural effusion, the concentration of PEDF measured by ELISA may not correctly reflect genuine loss or gain of PEDF in the effusions. Therefore, PEDF level was adjusted according to total protein in each pleural effusion.

Protein profiles of malignant and transudative pleural effusions

The laboratory characteristics of both types of pleural effusion are shown in table 1⇓. The protein profiles of 27 effusion samples were analysed by 2-DE with a pH range of 3–10 for greater protein separation. Figure 1a⇓ shows representative 2-DE images of proteins from one sample. In gels stained with Sypro Ruby, ∼643 spots were visualised using PDQuest image analysis software. By comparing the protein profiles of malignant and transudative effusions on 2-DE gels, seven protein spots were found showing a significant degree of differential expression (figs 1b–e⇓ and 2⇓).

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Fig. 1—

a) Representative two-dimensional gel electrophoresis of transudative pleural effusion with Sypro Ruby staining; and b–e) corresponding enlarged detail in: b, d) transudative and c, e) malignant pleural effusions. Spots 13, 14, 15, 26 and 27 (lower box in a (d and e)) and 53 and 54 (upper box in a (b and c)) represent individual proteins showing differing expression between transudative and malignant pleural effusions. Each of the protein spots are identified in table 2⇓.

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Fig. 2—

CyDye-stained two-dimensional electrophoresis (2-DE) gels showing selected proteins from a, d) transudative b, e) malignant effusions and c, f) merged images. The images were cropped from a representative 2-DE gel. g–l) Three-dimensional views of spots: g, h) 27; i, j) 15; and k, l) 54, from: g, i, k) transudative; and h, j, l) malignant effusions. The orange fluorescence in c) indicates greater expression of spots 13, 14, 15, 26 and 27 in the transudative effusion (Cy5). f). Similar results are seen for: spots 13, 14, 15 and 26, which correspond to fibrinogen γ-chain precursor; spots 53 and 54, which correspond to fibrinogen β-chain precursor; and spot 27, which corresponds to pigment epithelium-derived factor.

Identification of differentially expressed protein spots in malignant and transudative pleural effusions

The seven protein spots were excised and digested in gel with trypsin; this was followed by MALDI-Q-TOF analysis. The sequence coverage of the identified proteins, by peptide mass fingerprinting, ranged 3–15%, depending on the size of the proteins and the amount present. These spots were identified as fibrinogen β- and γ-chain precursors and PEDF (table 2⇓). The locations of these seven differentially expressed protein spots were marked with numbers in one representative gel, shown in figure 1⇑.

Pigment epithelium-derived factor levels in pleural effusions

The identification of spot 27 as PEDF was further confirmed by nanoflow LC-MS/MS. Since PEDF is the most potent natural anti-angiogenic factor, a decrease in PEDF concentration suggests that its low expression may play an essential role in mediating malignant effusion. In order to further evaluate the potential clinical significance of PEDF expression in pleural effusions, levels of PEDF were studied in all samples by Western blot analysis and ELISA. PEDF expression, as determined by Western blot analysis, was significantly lower in all malignant pleural effusions, irrespective of cancer type, than in transudative effusions (fig. 3⇓). The normalised concentration of PEDF was also significantly lower in malignant effusions (1.76±0.26 ng·mg protein ^-1; n = 14; p<0.0001) than in transudative effusions (4.33±0.52 ng·mg protein ^-1; n = 13) (fig. 4⇓).

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Fig. 3—

Western blot analysis revealing higher level of expression of pigment epithelium-derived factor in transudative (a) than in malignant effusions (b). Lane numbers correspond to subject number. c) Densitometric analysis of the Western blots confirmed this result. Horizontal bars represent means. AU: arbitrary unit. ^#: p<0.0001.

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Fig. 4—

Pigment pithelium-derived factor (PEDF) concentrations normalised to total protein were significantly higher in transudative (4.33±0.52 ng·mg protein ^-1; n = 13) than in malignant effusions (1.76±0.26 ng·mg protein ^-1; n = 14). Horizontal bars represent means. ^#: p<0.0001.