Thirty pairs of non-cancerous and cancerous pancreatic tissues were obtained from 30 patients (Table I) who underwent resection of pancreas with diagnosis of pancreatic cancer at the Department of Surgery II, Yamaguchi University Hospital.

None of the patients received any preoperative therapy. Written informed consent was obtained from all patients before surgery. Tissues were obtained immediately after surgery and stored at −80°C until use. The study protocol was approved by the Institutional Review Board for Human Use of the Yamaguchi University School of Medicine. The tissues were homogenized in lysis buffer (1% NP-40, 1 mM sodium vanadate, 1 mM PMSF, 10 mM NaF, 10 mM EDTA, 50 mM Tris, 165 mM NaCl, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) on ice (5). Suspensions were incubated for 2 h at 4°C, and the supernatants were stored at −80°C until they were used as samples. Ten pairs of samples were used for 2-DE, and twenty pairs for western blotting.

As the first dimension, isoelectric focusing (IEF) was conducted in an IPGphor 3 IEF unit (GE Healthcare, Buckinghamshire, UK) on 11-cm and pH 3–10 linear gradient IPG strips (Bio-Rad, Hercules, CA, USA) at 50 μA/strip. Protein (80 μg) was used for each 2-DE. Samples were mixed with 200 μl of rehydration buffer [8 M urea, 2% CHAPS, 0.01% bromophenol blue, 1.2% Destreak reagent (GE Healthcare)] and 0.5% IPG buffer, and loaded in the IPGphor strip holder. The strips were then focused by the following program: rehydration for 10 h (no voltage); 0–500 V for 4 h; 500–1,000 V for 1 h; 1,000–8,000 V for 4 h; 8,000 V for 20 min; and the final phase of 500 V from 20,000–30,000 Vh (6). After IEF, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a precast polyacrylamide gel with a linear concentration gradient of 5–20% (Bio-Rad) (7). The IPG strips were first equilibrated in equilibration buffer 1 (6 M urea, 0.5 M Tris-HCl, pH 8.8, 30% glycerol, 2% SDS, 2% 2-ME) for 10 min, and further in equilibration buffer 2 (6 M urea, 0.5 M Tris-HCl, pH 8.8, 30% glycerol, 2% SDS, 2.5% iodoacetamide) for 10 min. The IPG strips were then transferred onto the gels, which were run at 200 V (8). Each sample was replicated three times to ensure protein pattern reproducibility.

The SDS-PAGE gels were fixed with 40% ethanol and 10% acetic acid for 2.5 h. The gels were then treated with a fluorescent gel staining, Flamingo™ Fluorescent Gel Stain (Bio-Rad), for 18 h (9). The stained gels were washed with Milli-Q water 3 times, for 5 min each. These experimental procedures were carried out on a shaker.

The gels were scanned by using the ProXpress 2-D Proteomic Imaging System (PerkinElmer, Waltham, MA, USA) and then analyzed by using the Progenesis SameSpots software (Nonlinear Dynamics, Newcastle, UK) following the user manual. After image analysis, the gels were stained with See Pico™ (Benebiosis Co., Ltd., Seoul, Korea) overnight (10). The selected protein spots that displayed different intensities were cut from the gels and subjected to mass spectrometry (MS) analysis.

The gel pieces were destained by rinsing three times in 60% methanol, 0.05 M ammonium bicarbonate, and 5 mM DTT for 15 min. The sample in the gel piece was reduced twice in 50% methanol, 0.05 M ammonium bicarbonate, and 5 mM DTT for 10 min. The gel pieces were dehydrated twice in 100% acetonitrile (ACN) for 30 min. Enzyme digestion was carried out with an in-gel digestion reagent containing 10 μg/ml sequencing-grade-modified trypsin (Promega Corporation, Madison, WI, USA) in 30% ACN, 0.05 M ammonium bicarbonate, and 5 mM DTT at 30°C for 16 h. The samples were lyophilized overnight with the use of Labconco Lyph-lock 1L Model 77400 (Labconco, Kansas, MO, USA).

The lyophilized samples were dissolved in 15 μl of 0.1% formic acid, and then analyzed by using the LC-MS/MS system. Peptide sequencing of identified protein spots was carried out by using LC-MS/MS with a Spectrum Mill MS Proteomics Workbench (Agilent Technologies, Palo Alto, CA, USA). Fifteen microliters of each sample was injected and placed into separated columns (Zorbax 300SB-C18, 75 μm, 150 mm, Agilent Technologies). The Agilent 1100 capillary pump was operated in the following conditions: solvent A, 0.1% formic acid; solvent B, ACN in 0.1% formic acid; column flow, 0.3 μl/min for primary flow, otherwise 300 μl/min; gradient, 0–5 min 2% B and 60 min 60% B; stop time: 60 min. Proteins were identified in the Agilent Spectrum Mill MS Proteomics Workbench against the Swiss-Prot protein database search engine (http://kr.expasy.org/sprot/) and MASCOT MS/MS Ions Search engine (http://www.matrixscience.com/search_form_select.html). Standards for induction of candidate proteins were set as follows: filter by protein score >10.0, and filter peptide by score >8 (percent scored peak intensity).

The samples were separated by electrophoresis with SDS-PAGE gels and then transferred onto PVDF membranes at 90 mA for 78 min. The membranes were blocked overnight with TBS containing 5% milk at 4°C (11). They were incubated with the primary antibody against vinculin (anti-vinculin mouse monoclonal antibody, Sigma, St. Louis, MO, USA; 1:10,000), α-enolase (anti-enolase goat polyclonal antibody, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:1,000) and actin (anti-actin goat polyclonal antibody, Santa Cruz Biotechnology, Inc.; 1:200). The membranes were incubated with the secondary antibody conjugated with horseradish peroxidase (1:10,000) for 1 h at room temperature after washing three times with TBS containing Tween-20 and once with TBS. The membranes were treated with the ImmunoStar^® LD chemiluminescent reagent (Wako Pure Chemical Industries Ltd., Osaka, Japan), and protein spots were detected by using the Image Reader LAS-1000 Pro (Fujifilm Corporation, Tokyo, Japan).

2-DE gels were treated with a fluorescent gel stain, and then differences in the spot intensities between the tissues from pancreatic cancer and non-cancerous were analyzed and quantified by using the Progenesis SameSpots software. The results are summarized in Table II. At least 260 protein spots were matched on each 2-DE gel. Six upregulated spots (spots 1–6) were displayed on 2-DE gel with cancerous tissues at >1.5-fold higher intensity (Fig. 1). The protein expression levels were elevated significantly (P<0.05) in cancerous tissues when compared to paired non-cancerous tissues (Fig. 2).

The samples were digested with trypsin and then analyzed by using LC-MS/MS system, which identified the six upregulated protein spots as calreticulin (spot 1), glutathione synthetase (spot 2), stathmin (spot 3), vinculin (spot 4), α-enolase (spot 5) and glyceraldehyde-3-phosphate dehydrogenase (spot 6). The spot numbers are the same as those in Fig. 1. MS/MS data of these proteins are summarized in Table II.

There are still no reports regarding overexpression of vinculin in PC and its importance for cell adhesion and migration (12,13). Twenty pairs of pancreatic cancerous and non-cancerous tissues were analyzed by western blotting with anti-vinculin antibody, and the different intensities of the bands between cancerous and non-cancerous tissues were analyzed by the Student’s t-test (Fig. 3A). The mean intensities of the bands of cancerous and non-cancerous tissue samples were 125.2 and 66.4, respectively (Fig. 3A). Four pairs of cancerous and non-cancerous tissues were used for western blotting, to demonstrate the upregulation of α-enolase (14) as a positive control in cancerous tissues, compared to non-cancerous tissues (Fig. 3B). The appearance of vinculin on the 2-DE gels was located by 2-D western blotting (Fig. 4).