Seroreactivities for melanoma-associated antigens

To determine the frequencies and specificity patterns of antibody responses to melanoma-associated antigens, we tested by Western blot analyses the reactivities of sera of 94 patients with melanoma (see Table S1 for clinical details of the cases) against protein extracts from the melanoma cell line M-NRT separated by 1-dimensional SDS-PAGE (Figure 1A–C). As controls, the sera of 9 healthy individuals (Figure 1D), and of patients with cutaneous lymphoma, pancreas carcinoma or visceral leishmaniasis, an infectious disease that is known to induce responses to autoantigens of the infected host, were tested (Figure 1E). Overall, the signals were relatively weak given the high serum concentrations of 1∶6 used for these Western blots. A large number of faint background bands were detected with the sera of the healthy controls and patients alike as illustrated with Figure 1F which shows the Western blots with serum of a healthy donor (Serum 103) and the sera of two melanoma patients (Serum numbers 7 and 18). The Western blots shown in Figure 1F were developed as individual strips in separate plastic bags at a dilution of 1 in 200 which results in a better definition of the bands but is not suited for comparative screening of large numbers of sera. The arrows to the left of the Western blot lanes indicate shared signals found with patient and control sera alike. The arrows to the right of the lanes are stronger and indicate unique bands seen only with sera of melanoma patients. Serum 18 was from the same patient from whose tumor the melanoma cell line M-NRT had been established. This Western blot, thus, documents the reactivity in the autologous combination of tumor cells and serum. All other sera were from different patients or healthy donors, thus displaying the reactivity in heterogeneous combination of tumor cells and serum. The antigens thus detected are, therefore, expected to be antigens shared between the tumors of the serum donors and the test tumor cells M-NRT. With nearly two-thirds of the sera of the melanoma patients prominent bands are detectable that are not found with the healthy control sera or that are much stronger than those in the controls (Figure 1). The numbers of such prominent antigens detected with the reactive sera ranged between 2 and 6 per patient serum. Their masses were between 21 and 90 kDa with the bulk of the stronger bands between 40 and 80 kDa. The patients whose sera were tested represent, with the exception of ocular melanoma, all forms of melanoma. The majority of the patients were at stage 3 or 4 of disease but some were at earlier stages (Table S1). The course of disease ranged from relatively slow progression to aggressive disease. Within this range of patients there is no correlation of the numbers of antigens targeted, and frequencies or pattern of seropositivity with the clinical conditions. A few stronger reactivities were seen with the sera of healthy controls and patients with lymphoma, pancreas carcinoma or Leishmaniasis. These antigens were different than those detected with the sera of melanoma patients. In summary, about two-thirds of the patients had developed prominently detectable antibody responses against melanoma-associated antigens. The serospecificity patterns, however, were heterogeneous with no antigen that induced responses in a majority of the patients.

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Figure 1. Pattern of seroreactivities of melanoma patients, healthy controls and patients with other diseases against the melanoma cell line M-NRT.

Total protein extract of the tumor cells were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the sera of the melanoma patients (Panels A–C), of healthy control donors (Panel D, sera 102–110) or patients with cutaneous lymphoma (Panel E, sera 96–98), pancreas carcinoma (Panel E, serum 101) or visceral leishmaniasis (Panel E, sera 111–113). The numbers atop of each lane represent the number of the sera and are used throughout this report. For comparison, melanoma serum 7 was included in all blots. The Western blot analyses were done with the sera at a dilution of 1/6 in a multiple channel blotting/Western blot developing chamber for panels A–E. The letters underneath the lanes indicated the combinations of sera for the multiple probing of the 2D Western blots shown in Figure 2 and summarized in Table 1. Panel F: High-resolution 1D Western blot for comparison of the seroreactivities of melanoma patients and a healthy donor against the melanoma cell line M-NRT. In contrast to the blots shown in panels A–E, the sera were applied 1/200 to isolated lanes from SDS-PAGE in sealed plastic bags which results in a better definition of the bands when compared to blots from multiple blotting devices. Serum 18 is autologous to tumor cell line M-NRT, serum 7 is from a different patient and serum 103 from a healthy donor. The arrows to the left of the lanes indicated antigen bands that are shared between the patients and the healthy controls and occur in every blot. The arrows to the right of the lanes indicated prominent antigen that are detected only by the sera of melanoma patients.

https://doi.org/10.1371/journal.pone.0005199.g001

Identification of melanoma-associated antigens that raise antibody responses

Direct identification of antigens from 1-dimensional SDS-PAGE gels is not possible because of the complexity of the protein mixtures at every position in the lanes and difficulties in exactly aligning Western blots with silver-stained gels. We, therefore, separated the proteins of the melanoma cell line M-NRT by 2-dimensional electrophoresis with a pH gradient of 3 to 10 in the isoelectric focusing gels. Nine replica gels were prepared, eight used for Western blot analyses (Figure 2, panels A–H) and one for silver staining (Figure 2). Each of the Western blot filter A through G were probed successively with the sera of 8 melanoma patients, 56 sera in all. These are all available sera with which prominent bands were detected by 1DE. Blot H was tested with the sera of 9 healthy controls. The use of multiple sera per Western blot is required to expose several antigen spots per blot which is essential for spot pattern recognition and alignment of silver-stained proteins with Western blot spots. The serum combinations for multiple probing were selected to allow good pattern definition for assigning Western blot spots with protein spots in the silver-stained gel, and are listed in Table 1 where the numbering of the sera corresponds to the numbering in Figure 1. The letters underneath the blot lanes in Figure 1 refer to the 2D Western blots in Figure 2.

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Figure 2. Seroreactivities of melanoma patients and healthy controls against antigens of the melanoma cell line M-NRT isolated by 2DE.

Total protein extracts of M-NRT cells were separated with an isoelectric focusing pH range of 3–10 in the first and SDS-PAGE in the second dimension, blotted onto nitrocellulose membranes and probed with the sera of the patients as listed in Table 1. Serum dilutions were 1/200. The blots shown in Panels A through G were probed successively with 8 different patient sera each, blot H was probed with the sera of 9 healthy controls. The arrows indicated the antigens that could be assigned to protein spots in the silver-stained gel show with Figure 3. The numbering for the antigens is used throughout this report.

https://doi.org/10.1371/journal.pone.0005199.g002

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Table 1. Serum combinations for 2-dimensional Western blot analyses and numbers of identified antigens.

https://doi.org/10.1371/journal.pone.0005199.t001

The antigen signals were weak but clearly detectable in the 2-dimensional Western blots (Figure 2). The blots display 26, 19, 21, 20, 24, 3 and 15 antigen spots in panels A through G, respectively, altogether 128 antigen spots. With the sera of the healthy controls, 4 antigen spots were detected (Figure 2H). The antigens concur in numbers and mass distribution with the antigenicity patterns shown in Figure 1. Fifty-eight of the antigens could be assigned to protein spots in the silver-stained gels, 17, 9, 4, 12, 9, 1, 4 and 2 for the blots in Figure 2A through H, respectively. The antigen-protein assignments were done by first aligning gel and Western blots by their geometry as defined with artificial marker proteins spotted on cardinal points of the gels and marker protein spots detected in the blots after Ponceau S staining. A number of prominent marker spots were mapped for confirmation by partial blotting and matching the corresponding Western blots and silver-stained gels. Then, the spot patterns in the local environments of the antigens were compared taking spot sizes and shapes into consideration. The assigned and identified spots are indicated with arrows and numbered in the Western blots and the corresponding silver-stained gel shown in Figures 2 and Figure 3. The protein spots corresponding to the assigned antigens were excised from the gel, destained and subjected to trypsin digest. The resulting tryptic fragments were analyzed by peptide mass fingerprint by MALDI-TOF-MS with MASCOT and PROWL analyses of the peak lists. Forty-six of the 58 assigned antigens could, thereby, be identified. Spots number 10, 12, 13 and 24 were found in 2 Western blots, spots 1 and 5 in 3, spot 2 in 4, spot 3 in 5 and spots 4 and 9 in 6. All other assigned antigens were found only once. The 46 antigen spots, thus, represent 18 different antigens.

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Figure 3. Identification of melanoma-associated antigens detected by sera of melanoma patients.

The protein spots that could be assigned to antigen spots in the Western blots (arrows here and in Figure 2) were excised from the gels and treated with trypsin. The resulting fragments were analyzed by mass spectrometry to identify the antigens. Eighteen different antigens were identified that were found in 46 different spots in the Western blots shown in Figure 2.

https://doi.org/10.1371/journal.pone.0005199.g003

As examples for the mass-spectrometric identification of the antigens, the fingerprint mass spectra for spot number 9 identified as galectin-3, spot number 1 identified as the gelsolin-like actin filament-capping protein MCP, spot number 4 identified as heat shock protein 60 (HSP 60) and spot number 28 identified as the elongation factor EF-Tu are shown in Figure 4A, B, C and D, respectively. The spectra for the other antigens are provided as supplementary materials (Figures S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17) as indicated in Table 2 together with the gene identifier numbers, protein-chemical parameter and the results of the mass-spectrometric identification for all antigens. In the cases where masses remained that could not be assigned to the identified proteins, a secondary peptide mass fingerprint analysis was done with these remaining masses. In no case could a second protein be identified indicating that the analyzed spots did not contain major contaminating proteins. The identified antigens can be grouped according to their cell biological functions into different categories. Three of the antigens are heat shock proteins (HSP60, HSP70 and HSP70 protein 9B), 7 are enzymes of the cellular metabolism (enolase I, dienoyl-CoA reductase, aldolase A, fumarate hydratase, aldose reductase, aconitase and lactate dehydrogenase), hnRNP-1 is a nuclear protein involved in RNA processing, EF-Tu an elongation factor in protein biosynthesis, MCP affiliated with the cytoskeleton and involved in cell migration, calumenin a calcium-binding protein involved in regulation of metabolic processes, VCP participates in the regulation of protein export and the organization of the Golgi apparatus, LAP3 and PSME1 are involved in protein metabolism, and galectin-3 is a lectin with specificity for galactose. The sequence coverage of the mass-spectrometric fingerprint analysis of HnRNP is somewhat low. However, HnRNP had been repeatedly identified from different gels with better sequence coverage so that we are sure of its correct identification. For all other antigens the sequence coverage is sufficiently high for unequivocal identification of the corresponding proteins. Galectin is extracellularly expressed and involved in various interactions with serum proteins, other cells and extracellular matrix, and variously implicated in cancer-related processes such as metastasis formation and invasiveness. Enolase I, although a cytosolic protein, has been reported to be exported and is found in the extracellular environment of some tumors. Of the identified antigens, galectin-3 and HSP60 most often induce antibody responses in melanoma patients followed by calumenin and enolase I. For HSP60 and calumenin seroreactivity was also detected in healthy donors tested.

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Figure 4. Peptide mass fingerprint spectra of antigens identified by mass spectrometry.

Panel A shows the mass spectrum of the tryptic fragments for antigen spot number 9 identified as galectin-3, panel B the respective spectrum for antigen spot 1 identified as the actin filament capping protein MCP, panel C antigen spot 4 identified as the heat shock protein HSP60 and panel D antigen spot 28 identified as the elongation factor EF-Tu. The peptide mass fingerprint spectra for the other 17 antigen spots are provided with the supplementary materials indicated in Table 2 together with the statistics of the mass-spectrometric identification of the proteins. Asterisks indicate the tryptic fragment masses matched to the database sequences of the proteins.

https://doi.org/10.1371/journal.pone.0005199.g004

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Table 2. Antigens recognized by sera of melanoma patients and identified by proteome serology.

https://doi.org/10.1371/journal.pone.0005199.t002

Tumor cells, cell lines and sera

The tumor cells were isolated from melanoma metastases by treating minced tissue with collagenase free of contaminating protease activities and DNAse, passaging the resulting suspension through a cell sieve to remove connective tissue, allowing the tumor cells to adhere to tissue culture plastic plates and wash off non-adherent cells. The tumor cells were harvested by scraping them off the surface for direct use. Melanoma cell lines were established from such primary cells by culturing in DMEM (Gibco, Heidelberg) with 10% FCS (Biochrome, Berlin) and pencillin/streptomycine (Gibco, Heidelberg) at 37°C with 8% CO₂. The cells were processed for electrophoretic analysis immediately after harvest from the tumor nodules or from cell cultures. The sera were collected from 94 patients with melanoma at different stages of disease and 9 healthy donors of the same average age. The study with human subjects had been reviewed and approved by the institutional ethics committee of the Charité – Universitätsmedizin Berlin (Si. 277, September 11, 2003). The materials were obtained and used with written informed consent of the donors.

Protein sample preparation

The cells were collected by centrifugation at 1,600×g for 10 minutes at RT, washed three-times with PBS and solubilized by a protocol adapted from Görg et al. [48] and Chan et al. [49] with lysis buffer (7 M urea, 2 M thiourea, 2.5% Triton X-100, 2% β-mercaptoethanol, 0.8% Pharmalyte pH 3.5–10 (LKB, Freiburg, Germany), 200 µM Pefablock® (Merck, Darmstadt), 1 µM pepstatin (SIGMA, Munich, Germany) and 10 µM leupeptin (SIGMA, Munich, Germany) by vortexing and sonication for 10 minutes in an ice cooled water bath. The cell extracts were incubated for one hour at room temperature (RT) with 4,000 U/ml benzonase (Merck, Darmstadt, Germany) to degrade nucleic acids and then centrifuged at 350,000×g at 15°C for 15 minutes. The supernatants were collected, incubated once again with Benzonase for 10 minutes at RT and cleared by ultracentrifugation as before.

Two-dimensional gel electrophoresis (2DE)

Isoelectric focusing (IEF) was run in immobilized pH-gradient gel strips (IPG strips 180 mm×3 mm; Pharmacia, Freiburg) with a pH range of 3 to 10 [48], [49]. About 100 µg protein, extract of 1×10⁶ cells in 350 µl solubilization buffer were loaded into an IPG strip by in-gel re-swelling overnight at RT under silicon oil in a nitrogen- and water-saturated atmosphere to prevent oxidation of the protein and drying of the gel strips. The loaded strips were rinsed, mounted on a cooled ceramic plate and connected to the electrodes via water-wetted paper bridges. The IEF was run at 20°C under silicon oil in a nitrogen- and water-saturated atmosphere with 0.15 mA per IPG strip and 50 V for 18 h, 150 V for 1 h, 300 V for 2 h, 600 V for 1 h, 3,500 V for 6.5 h and 5,000 V for 3 h, a total of 40,000 Vh for pH 3–10 IPG strips. After the run, the IPG-strips were stored at −20°C. For the second dimension, IPG strips were thawed, rinsed with de-ionized water and equilibrated to SDS-PAGE conditions for 15 minutes in 6 M urea, 30% glycerol, 2% sodium dodecylsulfate (SDS), Tris pH 6.8 and bromophenol blue, 1% dithiothreitol (DTT) followed by 15 minutes in the same buffer but with 4% iodoacetamide instead of DTT. The equilibrated IPG-strips were rinsed with de-ionized water and placed gel-side to gel-side onto the 4.8% acrylamide, 0.13% bisacrylamide stacking gel of a horizontal SDS polyacrylamide gel with a 12.3% acrylamide, 0.34% bisacrylamide separation gel. The settings for the runs were 1,000 V, 40 W and 20 mA for 2–3 h for the pre-run to transfer the protein from the IPG strip into the SDS polyacrylamid gel, followed by 1,000 V, 40 W and 40 mA for the separation until the running front reached the anodic end of the gel.

Protein staining

The gels were stained with the high-sensitivity silver staining approach by Blum and colleagues [50]. Briefly, SDS-PAGE gels were fixed in 40% Methanol, 10% acetic acid for one hour or overnight. Then, the gels were washed three times for 20 minutes in de-ionized water, sensitized for one minute in 0.02% sodium thiosulfate, washed three times for 20 seconds in water and incubated for 20 minutes in silver staining solution (0.2% silver nitrate, 0.0074% formaldehyde). After washing three times for 20 seconds in water, the gels were incubated in developing solution (6% sodium carbonate, 0.00015% formaldehyde, 0.0004% sodium thiosulfate) until protein spots became visible. The reactions were stopped with 0.025% EDTA in water.

Western blots

The proteins from unstained SDS-PAGE were transferred onto nitrocellulose membranes (Schleicher & Schüll, Dassel, Germany) by semi-dry blotting for 2 hours at 400 mA. Free binding sites on the membranes were blocked with 5% low fat milk powder in Tris-buffered saline (TBS) for one hour at room temperature or at 4°C overnight. After blocking, the membranes were incubated with patient sera at a 200-fold dilution for one hour at room temperature, washed thrice for 10 minutes with TBS and incubated for 30 min with alkaline phosphatase-labeled anti human IgG (Anti-Human Ig-AP, Fab fragment; Boehringer Mannheim) at a 5,000-fold dilution. After washing three times 10 minutes with TBS, the membranes were equilibrated to developing buffer (100 mM NaCl, 100 mM Tris-HCl, pH 9.5) and developed in the dark with 100 µl BCIP and 100 µl NBT in 100 ml developing buffer until antigen spots became visible. The reactions were stopped by replacing the developing solution with water. For 1-dimensional Western blots, the proteins of the melanoma cells were separated by SDS-PAGE, 12% acrylamide, 0.8% bisacrylamide, blotted 1 h with 60 mA onto nitrocellulose and processed as above. For detection of antigens, the sera were applied in a multiple channel device (Figure 1A–E) or, after cutting the blots into strips, in individual sealed plastic bags (Figure 1F). Since efficient agitation of the serum solutions is not possible in the multiple channel device and antigen binding is solely dependent on diffusion, the sera had to be employed at a high 6-fold dilution. For the blots developed as strips in individual plastic bags, 200-fold dilutions of the sera were used as for the 2D Western blots.

Matching antigens and protein spots

To match antigen spots in Western blots with the corresponding protein spot in the silver-stained gel, the coordinates of the blots and the gel were defined, first, with artificial spots at the corner points, Ponceau S staining of the blot filter and aligning the spot pattern with the spot pattern of the silver-stained gel and partial blotting of a master gel and definition of marker spot sets, second, definition of spot pattern in the local environments of the antigen spots to match blots and gels in these local regions accurately, third, comparing the sizes and shapes of the antigen and protein spots and considering only those that are alike in these two parameter.

In-gel digestion of protein

The protein spots were excised manually with a self-made spot picker and de-stained as described by Gharadaghi and colleagues [51] with 50 µl of Farmer's reducing solution (15 mM potassium ferricyanide and 50 mM sodium thiosulfate, both dissolved in water), then washed three times for 5–10 minutes with 150 µl water. Then the gel spots were soaked in acetonitrile and dried under vacuum. The gel pieces were re-swollen in 7.5 µl of 5 mM ammonium bicarbonate with 75 ng of modified porcine trypsin (sequencing grade, modified; Promega; Madison, USA) to fragment the protein. After 10 minutes, 7.5 µl of 5 mM ammonium bicarbonate were added and the solution with the gel pieces incubated for at least 4 hours in a 37°C water bath. For MS analysis, 1.5 µl of the aqueous supernatants were mixed with 1 µl of 2,5-dihydroxybenzoic acid (DHB) (SIGMA, Munich, Germany) (5 mg/ml water) directly on MALDI targets (MTP AnchorChip 600/384, Bruker Daltronik, Bremen) and air-dried.

Mass spectrometry

Mass spectrometric measurements were made with a Reflex IV MALDI-TOF mass spectrometer (MS; Bruker Daltonik, Bremen) in reflector mode at an acceleration voltage of 20 kV. The MS was calibrated either with the external standards angiotensin II (1046.5 Da), angiotensin I (1296.6 Da), bombesin (1619.8 Da), substance P (1347.7 Da), ACTH 1–17 (2093.0 Da) and ACTH 18–39 (2465.1 Da) or with the autolytic 842.50 Da and 2211.10 Da trypsin fragments as internal standards. Monoisotopic peptide masses were recorded. The spectra were processed by the “Xmass” software (Bruker Daltonik, Bremen) and the peaks annotated automatically and checked manually. Post-source decay (PSD) analyses were done in 10 sections for the entire mass range and data accumulated from up to 300 shots per section. The peak lists of the mass spectra were the basis for peptide mass fingerprint analyses with the Mascot software (Matrix Science; http:www.matrixscience.com/search_form_select.html) and profound (prowl; http://prowl.rockefeller.edu/profound_bin/WebProFound.exe) using the NCBI sequence database.