Tissue samples from five patients (one male and four females) with a mean age of 56.8 years (range, 40–65 years) were analyzed in this study (Table 1). Tissues were collected between May 2009 and July 2011 at the Department of Otorhinolaryngology, Kaohsiung Medical University Hospital. Informed consent was obtained from all participants. The surgically-removed cholesteatoma specimens were resected from all patients during surgery, and retroauricular skin samples of the patients were also collected as controls.
Protein extraction and two-dimensional gel electrophoresis
Each sample (1 mm × 1 mm × 1 mm) was homogenized with 50 mM Tris-HCl buffer (pH 8.0, 10 mM EDTA and 10 mM protease cocktail) and centrifuged at 15,000 × g for 30 min. The proteins in the sample were then precipitated from the supernatant by adding 3 × volume of 10% TCA/acetone solution (containing 20 mM DTT) overnight at -20 °C. After centrifugation at 7000 × g for 30 min at 4 °C, the pellet was washed with cold acetone (containing 20 mM DTT), followed by air-drying using a Speed Vac (Branstead, Ipswich, England). The protein sample was then resuspended in sample buffer (6 M urea, 2 M thiourea, 0.5% CHAPS, 0.5% IPG buffer, 20 mM DTT, 0.002% bromophenol blue) at 4 °C overnight, and the protein concentrations measured using a 2-D Quant Kit (GE Healthcare, Chicago, IL, USA).
First-dimension electrophoresis was carried out using a GE Healthcare Ettan IPGphor 3, as previously described(17). Proteins (250 ∝ g) extracted from cholesteatoma and retroauricular skin tissues were loaded on 11-cm IPG strips (pI 4–7 and pI 3–11, Immobiline DryStrip) for first-dimension electrophoresis, followed by separation on sodium dodecyl sulphate polyacrylamide electrophoresis gel (12.5%) using an SE 600 Ruby (Hoeffer) at 150 V for 6.5 h.
Protein spot identification by LC-MS/MS
Protein spots on the 2-DE gel were visualized after CBR staining. Spots of interest were then excised into a piece (1 mm × 1 mm) from the 2-DE gel and digested by trypsin. The in-gel digested sample was identified by LC-MS/MS using an AB SCIEX QTRAP 5500Q mass spectrometer (Applied Biosystems, CA, USA). The detailed procedure was described in a previous study(17).
Western blot analysis
1-DE or 2-DE PAGE samples were transferred to a PVDF membrane (Millipore) under 0.4 A for 2 h using Transphor TE 62 (GE Healthcare, Chicago, IL, USA). The membrane was first incubated with rabbit anti-human CRT and AnxA2 (ProteinTech Group, Chicago, IL, USA) or rabbit anti-human β-actin antibodies (Sigma, St. Louis, MO, USA), then with goat anti-rabbit or rabbit anti-mouse horseradish peroxidase-conjugated IgG (1/5,000 dilution, Millipore, Bellerica, MA, USA) at room temperature for 2 h. After washing with PBST three times, the protein concentrations were determined from the chemiluminesence intensity using Pierce™ ECL Western blotting reagents (Thermo Fisher Scientific, Waltham, MA, USA).
Immunohistochemical staining and immunostaining evaluation of calreticulin
The tissue samples of the five patients analyzed in the proteomics study were also used for immunohistochemical analysis. A cholesteatoma specimen and a retroauricular skin specimen of each patient were prepared as 4-µm-thick paraffin sections and subjected to immunohistochemical analysis. After de-paraffinization, rehydration and autoclave treatment at 121 °C for 10 min in DAKO Target Retrieval Solution, pH 9.0 (DAKO, Glostrup, Denmark) to induce antigen retrieval, the paraffin sections of all samples were incubated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. The sections were then stained with CRT or AnxA2 polyclonal antibodies (1:50; ProteinTech Group, Chicago, IL, USA) at room temperature for 1 h, and a DAKO REAL EnVision Detection kit (DAKO, Glostrup, Denmark) was then used as the secondary antibody. After incubation in 3’3-diaminobenzidine for 5 min, the sections were counterstained with Mayer’s hematoxylin and mounted. Negative controls were prepared by replacing the primary antibody with non-immune serum of the same species. They were assigned a score number according to the following rules: a score of 0 for 0% epithelium cells positive, a score of 1 for 1–24% epithelium cells positive, a score of 2 for 25–49% epithelium cells positive, a score of 3 for 50–74% epithelium cells positive and score of 4 for 75–100% epithelium cells positive. The intensity of cellular staining was also assigned a score number: a score of 0 for zero intensity, a score of 1 for weak intensity, a score of 2 for moderate intensity and a score of 3 for strong intensity(17). A staining score was obtained by multiplying the percentage score with the intensity score, with a maximum score of 12. Statistical evaluations were performed using the paired t-test. A difference considered statistically significance is 0.05.