A total of 15 freshly collected serum specimens were obtained from patients (mean age, 51.70±3.10 years; all male) diagnosed with PC (without hypertension or diabetes) between January 2013 and January 2014 at Shanxi Provincial Cancer Hospital (Taiyuan, China). The samples were obtained prior to treatment (Fig. 1). The diagnosis criteria were based on the Guidelines for Diagnosis and Treatment of Pancreatic Cancer (2014 Edition) from Group of Pancreatic Surgery, Branch of Surgery, Chinese Medical Association (11). The American Joint Committee on Cancer (AJCC) Tumor-Node-Metastasis (TNM) staging is the most widely used cancer staging system worldwide. According to the AJCC 8th edition staging system for patients with PC, 7 patients had been diagnosed as stage IIB, and 8 patients had been diagnosed as stage III (12) (Table I). The tumor status of 15 patients with PC are shown in Table II. During the same period, 10 healthy control subjects undergoing physical examinations at Shanxi Provincial Cancer Hospital (mean age, 56.40±2.42 years; all male) were enrolled. The present study was approved by The Ethics Committee of Shanxi Provincial Cancer Hospital (approval no. 201732), and written informed consent was obtained from each participant.

Blood samples were collected in the morning before the patients had eaten, placed at 4°C for 1.5 h, and centrifuged at 3,000 × g for 10 min at 4°C. The resulting supernatant (serum) was collected and stored at −80°C.

The serum samples from 15 patients and 10 healthy controls collected at baseline were immuno-affinity depleted of highly abundant proteins using a Multiple Affinity Removal Column Human 14 (4.6×50 mm; Agilent), according to the manufacturer's protocol. Lowly abundant proteins were collected on a high-performance liquid chromatography (HPLC) system (1260 Infinity II LC System; Agilent) using dilution, stripping and neutralization buffers provided, according to the manufacturer's protocol. The detailed conditions were: Column, Agilent HC-C18 (4.6×150 mm, 5 µm); mobile phase, methanol/water 20:80 (v/v) containing 0.05 M potassium dihydrogen phosphate (pH 3.5); detection wavelength, 238 nm; flow rate, 1.0 ml/min; column temperature, 35°C; injection volume, 20 µl. To 200 µl plasma, 600 µl 10% perchloric acid was added; after mixing, the sample was centrifuged at 16,000 × g for 10 min on a GL-20G-II high-speed desktop refrigerated centrifuge (Flying Pigeon; Shanghai Anting Scientific Instrument Factory, Shanghai). Following this, 20 µl of the resulting supernatant was used for quantitative analysis.

The serum original samples of patients with pancreatic cancer and healthy controls were analyzed by SDS-PAGE. Serum protein levels were determined using a Bradford assay kit (Beyotime Institute of Biotechnology). The depletion efficiency of each approach was evaluated by SDS-PAGE. A Coomassie Blue Stain kit PH0351 (Feijing Scientific Research Reagent Store) was used containing 100 ml Coomassie Blue stain and 500 ml Coomassie Blue decoloring solution. Coomassie blue staining was performed at 23°C for 2–4 h followed by decolorizing at 23°C for 4–8 h. The type of gel was SDS-PAGE gels. No two-dimensional separation was used. Then, equal amounts (15 µg) of total protein were separated by 12% SDS-PAGE electrophoresis.

In total, 200 µg of highly abundant protein depleted serum was denatured with 200 µl dithiothreitol (20 mmol/l) at 37°C for 1 h, and alkylated with iodoacetamide (50 mmol/l) at room temperature for 45 min. Urea (8 mol/l) was used for elution, and ammonium bicarbonate (25 mmol/l) for washing. Finally, trypsin was used overnight to digest the serum proteins at 37°C, and the reactions were terminated by addition of formic acid at a final concentration of 1%. Next, iTRAQ labeling was performed for peptides from healthy controls and PC samples using an iTRAQ reagent 8-plex kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Peptides were desalted, dried and labeled with different isobaric tags for 2 h at 25°C. Peptides enriched in serum samples from healthy controls and patients with PC were labeled with 116 and 117 tags, respectively. Finally, the labeled peptides were dried under vacuum and stored at −80°C.

Digestion and iTRAQ labeled serum samples were reconstituted in 400 µl of 20 mm ammonium formate/2% acetonitrile (pH 10). Following this, the samples were loaded on an RP C18 capillary column (3, 4.6 µmx250 mm; Shimadzu Corporation) and fractionated on an Agilent 1100 series HPLC instrument by basic RP chromatography at a flow rate of 1.0 ml/min. The mobile phase consisted of 90% acetonitrile (pH 10), used with a gradient of 5 to 30% solvent B (formic acid), for 0–60 min. Fractions were collected every 1 min for a total of 60 fractions; early and late fractions were pooled, resulting in a total of 30 fractions. The pooled samples were reconstituted in 0.1% formic acid for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Peptides in each fraction were separated on an RP C18 capillary column (3, 75 µmx100 mm) and eluted using a linear gradient of 5 to 30% solvent B1 (0.1% v/v) formic acid in acetonitrile for 40 min, at a flow rate of 300 nl/min). MS data were acquired by the shotgun proteomics method (LTQ-Orbitrap mass spectrometer; Sanofi S.A.); in each cycle, a full scan was acquired at a resolution of 30,000 dpl with a mass range of 380–1,600 m/z. Up to 10 of the most intense precursor ions with charge range of +2 to +4 were selected with an isolation window of 2 Da, and subsequently fragmented by higher-energy collisional dissociation with a normalized collision energy of 40%. The MS/MS scans were acquired at a resolution of 7,500 dpl. Precursor ions were dynamically excluded from reselection for 60 sec.

Raw data from the mass spectrometer were analyzed by the Mascot (version 2.3.02; http://www.matrixscience.com/) and Scaffold (version 4.4.3; Proteome Software, Inc.) programs. The search database was Swiss-Prot (uniprot.org) (human species). Tyrosine served as a variable modifier protein. The search parameters were: Trypsin allowed P enzyme specificity, with up to two missing cleavages; precursor ion mass tolerance, ±10 ppm; fragment ion mass tolerance, 0.05 Da; fixed modification, carbamidomethylation and no variable modification. The identified proteins were quantified according to the iTRAQ of the specific polypeptide. Differential proteins in serum proteome: Differential multiple >1.5, P<0.05.

Differentially expressed proteins (ratio >1.5 or <0.5; P<0.05) were imported into the Panther database (pantherdb.org/) and matched with the human genome data. Following this, the proteins were classified into molecular function, biological process and cellular component according to gene ontology (GO). Canonical pathway, upstream regulation and protein-protein interaction analyses were performed by the IPA software (Ingenuity Systems; Qiagen, Inc.).

Data are presented as mean ± standard deviation. SPSS 19.0 software (SPSS; IBM Corp.) was used to analyze the results. Student's t-test was applied to compare mean values between the two groups. P<0.05 was considered to indicate a statistically significant difference.

As presented in Table III, serum CA19-9, CA242, CA724, CA50 and VEGF levels in patients with PC were significantly increased compared with the values of healthy controls. In addition, these diagnostic factors were higher than normal levels, except VEGF.

Serum samples from patients with PC and control patients were immuno-affinity depleted of the 14 most abundant proteins using Multiple Affinity Removal Column Human 14, and SDS-PAGE was performed to assess various serum samples in the two patient groups. As presented in Fig. 2, low abundance proteins demonstrated improved separation and exhibited higher concentrations. In addition, an increased number of low abundance proteins was observed; however, the albumin band disappeared. In the serum proteome, 251,479 spectra were obtained. Proteomics revealed 531 proteins, of which 442 were quantifiable.

Based on the aforementioned 442 quantifiable proteins, more than two peptide segments were identified by Mascot and Scaffold. There were 76 differentially expressed proteins in samples from patients with PC compared with control specimens (Table IV). Among them, 70 proteins were downregulated, with 24 demonstrating a coordination ratio of 0.4 or more. Meanwhile, six proteins were upregulated by 2–3 fold. To comprehensively assess the biological significance of the 76 differentially expressed proteins, analysis was performed by the Panther gene classification system, which includes biology processes, cellular components, molecular functions and protein classes. Compared with the normal serum proteome database (pantherdb.org/chart/summary/pantherChart.jsp?filterLevel=1&chartType=1&listType=1&type=5&species=Homo%20sapiens), proteins associated with the extracellular matrix, receptor activity and enzyme regulator activity were upregulated in patients with PC, and those involved in the extracellular matrix accounted for 6.8% of all proteins (Fig. 3). However, proteins involved in nucleic acid binding transcription factor activity and metabolic processes were significantly downregulated.

A bioinformatics analysis of the identified differentially expressed proteins was performed using the IPA software. As presented in Fig. 4, certain pathways were significantly altered. Proteins in the coagulation system (P=4.97; ratio, 1.43×10⁻¹) and lipopolysaccharide/interleukin-1 mediated inhibition of retinoid X receptor (RXR) function (P=3.98; ratio, 4.11×10⁻²) were significantly upregulated. In comparison, liver X receptor/RXR activation (P=13.00; ratio, 1.24×10⁻¹), production of nitric oxide and reactive oxygen species in macrophages (P=9.38; ratio, 7.78×10⁻²), paxillin signaling (P=2.78; ratio, 4.95×10⁻²), and integrin signaling (P=2.14; ratio, 2.99×10⁻²) were significantly downregulated. In addition, some differentially expressed proteins were involved in cell survival, molecular translocation, viral infection and lipid efflux, revealing decreased levels. A protein interaction network analysis also revealed that certain associated proteins were upregulated or downregulated (Fig. 5).