A proteomics approach was used to ascertain whether the UVB generated OS determined a qualitative and/or quantitative modification in the NHEK protein profiling. The UVB dosage chosen (20 J/m²) was able to induce intermediate cell damage without suppressing the cell response mechanisms (see Additional file 1). Total proteins extracted from UVB-irradiated and from control cells were subjected to two dimensional gel electrophoresis (2-DE). Software-assisted densitometric analysis of resolved gels allowed a comparison of the respective protein repertoires and the determination of quantitative modifications in the UVB-irradiated cells as compared to non-irradiated ones. Representative Coomassie-stained gels are shown in Figure 1, panel a and panel b.

The overall 2-DE pattern of UVB-treated cells and control cells were similar. However 15 spots were found to be differentially expressed with at least 1.5-fold increase or decrease (p < 0.059) compared to control cells. To assess reproducibility, the correlation coefficient among six replicated gels was calculated; the average r value of 0.9 indicated a high quality 2-DE gels and good reproducibility of culture and treatment conditions.

Figure 2 shows an enlarged image of 2-DE gel of UVB-treated NHEK, with the differential expression of protein spots highlighted by circles and numbers.

The spots of differentially expressed proteins were excised from the gels, proteolysed and subjected to MS analysis. The database search with data deriving from Peptide Mass Fingerprint MALDI-ToF experiments allowed the identification of the spots. The list of the identified proteins is reported in Table 1, together with their quantitative variations, expressed as fold compared to control. 12 proteins were found to have higher abundance levels in UVB-irradiated NHEK compared to the control group, while 3 proteins were found to have a lower abundance level. All listed proteins included in Table 1 had p-values = 0.05, good sequence coverage, significant protein scores and similar observed and calculated molecular weights and isoelectric points.

Carbonylation is the most widely studied oxidative modification of proteins because of its ease in detection by the Western blot. Indeed, the protein-bound carbonyl groups upon reaction with DNPH generate stable protein-hydrazone complexes which are then easily detected by specific antibodies. The specific carbonylation level of each single spot was evaluated through the ratio between the carbonyl level of a protein spot on the nitrocellulose membrane and the protein level of its corresponding protein spot in the gel - assessed by Bio-Safe Coomassie staining and image analysis - and expressed as carbonyl level per unit of protein. In Figure 3 two-dimensional carbonyl immunoblots from control and UVB-treated NHEK are shown. 7 proteins resulted to be significantly more oxidized in irradiated cells compared to control cells. Table 2 illustrates the list of proteins that were successfully identified by the mass spectrometry, along with protein scores, sequence coverage, pI, Mw values and the increase of specific carbonyl levels.

To verify the proteomics and redox proteomics results, validation studies on protein up- or down-regulation and protein carbonylation were performed.

The modulation of protein expression level was validated by WB analysis in the case of glucose-regulated protein 78 (GRP78) and HSP70. The results are shown in Figure 4, where an increase of HSP70 and a parallel decrease of GRP78 can be seen in irradiated cells, thus confirming the same behaviour detected by proteomics, i.e. GRP78 down-regulation and HSP70 up-regulation.

Redox proteomics results were validated by WB immunochemical detection of carbonylated proteins. In Figure 5, the carbonyl levels of protein disulfide isomerase A3 (PDI A3), Annexin 2 (Anx2) and GRP78 in UVB-treated cells were respectively about 180%, 160% and 320% in comparison to those of control cells, thus qualitatively confirming the redox proteomics findings.

The increase of carbonyl levels of PDI A3, Anx2 and GRP78 in UVB-treated cells were more robust when detected by the proteomics method. The differences in the magnitude of fold changes of carbonyl levels between the two techniques are likely because proteomics measures the carbonyl level per unit of protein, whereas WB measures the carbonyl level of the total protein.

To evaluate the redox status of PDI A3 in NHEK cells, before and after treatment with UVB radiation, the cells were treated with the membrane permeable alkylating agent NEM to prevent disulfide exchange and freeze redox status. Then, cell lysates were treated with a second larger alkylating agent (AMS) that causes a shift in mobility when the protein is separated by SDS-PAGE. NEM alkylation performed on intact cells prevents AMS modification of free thiol residues present in the proteins. The second step in the alkylation was carried out after treating cell lysates with a thiol reducing agent that allows the modification of protein thiol residues present as disulfide bonds in intact cells. Thus the oxidized form of a protein can be resolved from the reduced one by its decreased electrophoretic mobility.

Our results showed that in control cells PDI A3 was present both in oxidized and reduced form. Accordingly, WB analysis with anti-PDI A3 antibody revealed the presence of two bands related to oxidized and reduced forms of PDI A3 in NHEK untreated cells (Figure 6 lane 1). The treatment of cells with UVB radiation resulted in a significant increase in the oxidized form of PDI A3 as revealed by mobility shift (Figure 6 lane 2).

Normal human epithelial keratinocytes (NHEK) were obtained from children's foreskins kindly donated by patients attending the general surgery division at the Ospedale S. Pertini (Rome, Italy), whose parents had released a full informed consent. NHEK were isolated and grown according to standard procedures [45] with minor modifications. Briefly, after scraping away excess fat and subcutaneous tissue, the foreskins were floated in 0.25% trypsin solution at 4°C overnight. The epidermis was then lifted off and placed in 10 ml of 0.5% trypsin-EDTA (Invitrogen Life Technologies, s.r.l. San Giuliano Milanese, Italy) at 37°C for 1 h under continuous mild stirring. Trypsin was neutralised by soybean trypsin inhibitor, the cell suspension was pelletted for 10 min at 200 × g, washed twice in phosphate buffered saline (PBS) and cultivated in keratinocytes serum free medium (K-SFM) (Invitrogen). Cells were maintained in a humidified incubator with 5% CO₂ at 37°C and passaged twice a week at such a density they never reached 80% confluency. To avoid bias of senescence modification in cell metabolism, cells between third and eighth passages were used in the present study. For UVB treatment, cells were plated in a 100 mm/petri dish at a density of 80,000 cells/cm² (i.e. 50% confluency). Immediately before irradiation, the medium was removed and the monolayers were exposed to UVB in a home-made irradiation hood. To prevent overheating of monolayer, dishes were seated on a water bath at 37°C during the whole irradiation. UVB source was provided by a bank of Sankyo Denki G15T8E fluorescent tubes emitting 270-320 nm wavelength with a peak at 313 nm. The energy actually incident onto the working area was measured by a UVX Radiometer (UVP Inc., Upland, CA) and expressed in J/m². The UV dosage of 20 J/m² (8 second exposure time) was chosen according to the results obtained in our previous studies [13] Such a dose was able to induce intermediate cell damage without suppressing the cell response mechanisms (see Additional file 1). After UV exposure, fresh medium was added and the cultures were further incubated. For negative control, cultures were decanted and placed in the irradiation chamber while keeping the UVB lamps switched off. Five hours after irradiation, cells were washed twice with ice cold PBS, scraped with a rubber policeman and pelletted.

Cell pellets were lysated in 10 mM HEPES buffer (pH 7.4) containing 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH₂PO₄, 0.1 mM EDTA, and 0.6 mM MgSO₄ as well as proteinase inhibitors: leupeptin (0.5 mg/ml), pepstatin (0.7 μg/ml), type II S soybean trypsin inhibitor (0.5 μg/ml), and PMSF (40 μg/ml). Cell lysates were centrifuged at 14,000 × g for 10 min to remove debris. Protein concentration in the supernatant was determined by using the Coomassie (Bradford) Protein Assay (Pierce, Rockford, IL, USA).

The analysis was performed as previously described [13]. Sample volumes equivalent to 150 μg proteins were precipitated by adding of 100% ice-cold trichloroacetic acid (TCA) to a final concentration of 15% and placed on ice for 10 min. Precipitates were centrifuged at 15,000 × g for 2 min. The pellets were washed three times with 0.5 ml ethanol/ethyl acetate 1:1 solution. After centrifugation and washing, the samples were dissolved with 200 μL of rehydration buffer (8 M urea, 20 mM dithiothreitol, 2.0% (w/v) CHAPS, 0.2% Biolytes, 2 M thiourea and bromophenol blue).

For the first-dimension electrophoresis, 200 μL of sample solution were applied to a ReadyStrip™ IPG strip pH 3-10 (Bio-Rad Laboratories S.r.l., Segrate, Milano, Italy). The strips were soaked in the sample solution for 1 h to allow the uptake of proteins. The strip was then actively rehydrated in a Protean IEF Cell Apparatus (Bio-Rad) for 16 h at 50 V. The isoelectric focusing was performed at 300 V for 2 h linearly; 500 V for 2 h linearly; 1000 V for 2 h linearly, 8000 V for 8 h linearly and 8000 V for 10 h rapidly. All the processes above were carried out at room temperature. The focused IEF strips were stored at -80°C until second dimension electrophoresis was performed.

For second dimension electrophoresis, thawed strips were equilibrated for 10 min in 50 mM Tris-HCl (pH 6.8) containing 6 M urea, 1% (w/v) sodium dodecyl sulfate (SDS), 30% (v/v) glycerol, and 0.5% dithiothreitol, and then re-equilibrated for 15 min in the same buffer containing 4.5% iodacetamide in place of dithiothreitol. Linear Gradient (4-12%) Precast criterion XT gels (Bio-Rad) were used to perform second dimension electrophoresis. Precision Protein™ Standards (Bio-Rad) were run along with the sample at 200 V for 65 min.

For expression analysis, after electrophoresis the gels were incubated 20 min in fixing solution (7% acetic acid, 10% methanol), stained for 1 h in approximately 40 ml of Bio-Safe Coomassie Gel Stain (Bio-Rad) under continuous gentle agitation and destained overnight in deionized water.

For the protein oxidation analysis, gels were transferred to nitrocellulose membrane (Bio-Rad) using Criterion Blotter apparatus (Bio-Rad) at 100 V for 1 h according to the supplier's instructions. The carbonyl levels were detected by post-Western blot derivatization of 2D nitrocellulose membrane [46]. Briefly, following the electroblotting procedure, the nitrocellulose membranes were equilibrated in 20% methanol for 5 min and then incubated in 2 N HCl for 5 min. Next, membranes were incubated in 0.5 mM 2,4-dinitrophenyl hydrazine (DNPH) solution for 5 min sharp. The membranes were washed three times in 2 N HCl and five times in 50% methanol (5 min each wash). The 2,4-dinitrophenyl hydrazone (DNP) adducts of the protein carbonyls were detected on the nitrocellulose sheet using a primary rabbit antibody (Millipore Corp., MA, USA) specific to DNP-protein adduct (1:100), followed by a secondary goat anti-rabbit IgG alkaline-phosphatase conjugated antibody (Sigma-Aldrich, Milano, Italy). The resultant stain was developed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) solution (SigmaFast tablets from Sigma).

The 12 gels (n = 6 controls and n = 6 UVB-treated cells) and 12 nitrocellulose blots were scanned and saved in TIF format using a Scanjet 3300C (Hewlett Packard). PDQuest 2D Analysis Software (version 7.2.0, Bio-Rad) was used for protein spot matching and analysis and to compare proteins and DNP immunoreactivity content between UV-treated and control cells. This software offers powerful comparative analysis and is specifically designed to analyze many gels or blots at once. Powerful automatching algorithms quickly and accurately match gels or blots and sophisticated statistical analysis tools identify experimentally significant spots. The principles of measuring intensity values by 2-D analysis software were similar to those of densitometric measurement. The average mode of background subtraction was used to normalize intensity values amount of protein (total protein on gel versus DNP-bound protein on the membrane) per spot. Once spots had been matched, images were manually edited to confirm proper spot detection and matching. The intensity of each protein spot was normalized as a percentage of total volume, corresponding to pixel intensity integrated over the area of each spot and divided by the sum of all spots in the gel to account for staining variability. Following manual editing and matching confirmation, average normalized spot volumes (pixel intensity over spot area) were compared between UVB-treated and control cells. Target candidates were identified as protein spots that changed at least 1.5-fold versus their specific control or alternatively that were either present or absent either in control or in experimental gel. Protein spots with greater than 50% internal variance were removed from the target list. Finally, remaining individual candidates were visually examined to ensure that the change was consistent in all gels.

After completion of spot matching, the normalized intensity of each protein spot from individual gels was compared between groups using statistical analysis. Statistical significance was assessed by a two-tailed Student's t-test, the method of statistical analysis most appropriate for proteomic analysis of small number of protein spots [47]. P values < 0.05 were considered significant for comparison between control and experimental data (UV-treated NHEK cells).

Selected spots were manually excised from gels and submitted to trypsin proteolysis, as described by Mignogna et al. [48], with little difference. In short, after four destaining steps using 5% (30 min), 50% (2 times, 30 min each), and 100% (10 min) acetonitrile in 25 mM ammonium bicarbonate, about 165 ng of trypsin (modified porcine variant, Promega, Madison, WI, USA) were solubilised in 15 μl of a 25 mM ammonium bicarbonate digestion buffer and added to each vacuum-dried gel spot. Digestion was performed at 37°C overnight. The peptide mixtures were eluted directly onto an appropriate MALDI target plate with 1.3 μl of α-cyano-4-hydroxy-trans-cinnamic acid matrix solution (2 mg/ml) in 70% acetonitrile containing 0.1% TFA (v/v). MALDI-ToF MS analyses were performed in a Voyager-DE STR instrument (Applied Biosystems, Framingham, MA, USA) equipped with a 337 nm nitrogen laser and operating in reflector mode. Mass data were obtained by accumulating several spectra from laser shots with an accelerating voltage of 20 kV. All mass spectra were externally calibrated using a standard peptide mixture containing des-Arg-bradykinin (m/z 904.4681), angiotensin I (m/z 1296.6853), 1-17 (m/z 2093.0867), and 18-39 (m/z 2465.1989) adrenocorticotropic hormone fragments. Two tryptic autolytic peptides were also used for the internal calibration (m/z 842.5100 and 2807.3145). Several ion signals were submitted to fragmentation by post source decay (PSD). PSD fragment ion spectra were obtained after isolation of selected precursor ions using a timed ion selector (TIS), performing 10 steps of the reflectron voltage; for each individual step the voltage was decreased 25% with respect to the previous step. The individual segments were automatically stitched together. The PSD fragment ions were measured as isotopically averaged masses. Calibration was performed with PSD spectra of angiotensin.

The MS and MS/MS data were analysed by MoverZ program (v. 2002, http://bioinformatics.genomicsolutions.com), according to default parameters.

Identification by peptide mass fingerprint (PMF), with the monoisotopic mass list obtained from each spot, after exclusion of expected contaminant mass values by Peak Erazor program http://www.protein.sdu.dk/gpmaw/Help/PeakErazor/peakerazor.html, was performed using the Mascot search engine (v. 2.1) against SwissProt database (v. 52.2, entries 263525). Up to one missed cleavage, 50 ppm measurement tolerance, oxidation at methionine (variable modification) and carbamidomethyl cysteine (fixed modification) were considered. Post-translational modifications were not considered. Identifications were validated when the probability-based Mowse protein score was significant according to Mascot [49].

Identification by tandem mass spectrometry analyses was performed using the Mascot search program (Version 2.1) against human SwissProt database (v. 54.6×, 290484 sequences; 107100015 residues; date 2008/01/03), with mass tolerance of ± 0.5 Da for the precursor ions and ± 0.8 Da for the fragment ions, with carbamidomethyl cysteine as fixed modification. The expectation value (E-value) for accepting identification by MS/MS spectra was set to < 0.1, with a default significance threshold p < 0.05, that provides a 95% confidence level.

For Western blot analysis a 40 μg aliquot of each protein sample was separated through a 12% SDS-PAGE and electroblotted (1 h at 100 V) to nitrocellulose membranes (Bio-Rad) using 25 mM Tris, 192 mM glycine and 20% (v/v) methanol. Equal protein loading was confirmed by 0.2% v/v Ponceau S in 7% acetic acid blot staining. Blotted membranes were blocked with 5% no-fat milk and challenged with appropriate primary antibodies, namely Anx2 mouse monoclonal IgG (Abnova GmbH, Heidelberg, Germany), GRP78 rat monoclonal IgG (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA) and PDI A3 antibody (kindly provided by Prof. F. Altieri, Rome) for 1 h at room temperature. Unbound antibodies were removed by washing it twice with Tris-buffered saline containing 0.1% Tween 20, for 5 minutes. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody diluted 1:5000. Protein bands were visualized with ECL PlusTM (Amersham, NJ, USA) according to the manufacturer's protocol.

The immunoprecipitation was performed as described by Lauderback et al. [50] Antibodies were added directly to cell lysates with IP Buffer (NaCl 0.15 M, NP-40 0.5%, Tris-HCl 50 mM pH 7.2, protease inhibitors) and the mixture was incubated on a rotary mixer overnight at 4°C. The antigen/antibody complexes were precipitated with protein-A-conjugated agarose beads if the antibodies were raised in rabbit or with protein G-conjugated agarose beads if the antibodies were raised in goat or mouse. Agarose beads were added in 50 μl aliquots from a stock of 300 mg/ml in PBS and mixed on a rotary mixer for 1 h at room temperature. Beads were then pelletted and washed three times with washing buffer (pH 8, 50 mM Tris HCl, 150 mM NaCl, 0.1% Tween 20). Proteins were eluted in IEF rehydration buffer followed by a 2D electrophoresis (Anx2) or in sample buffer for post-Western blot analysis (PDI A3, Anx2 and GRP78).

To determine the in vivo redox status of PDI A3/ERp57, UVB-treated NHEK were subjected to thiols sequential alkylation with N-ethylmaleimide (NEM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), as described by Jessop and Bulleid [51] and modified according to Kim-Han and O'Malley [52]. In short, cells were incubated with 25 mM NEM to block free thiols and then lysed in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail. Lysates were then treated with 2% SDS and 50 mM DTT in order to reduce all thiol residues, precipitated with 10% trichloroacetic acid and washed with 70% ice cold acetone. Finally, proteins were resuspended in 80 mM Tris-HCl pH 6.8, 2% SDS, protease inhibitor cocktail and 30 mM AMS in order to alkylate free thiol residues. Samples were separated by 10% SDS-PAGE and analyzed by Western blot with PDI A3 specific antibody [51,52]. AMS alkylated proteins (i.e. proteins with oxidised thiol groups) had a reduced electrophoretic mobility compared with non derivatized (reduced) proteins.

Two-sided, Student's t-tests were used to analyze differences in protein levels between UVB-treated NHEK cell lysates and control untreated lysates. According to the exhaustive analysis of Maurer and Peters [47] the significance of carbonylation change of specific proteins was evaluated via nonparametric Mann-Whitney-Wilcoxon test. P < 0.05 was considered statistically significant.