Mass spectrometry‑based iTRAQ analysis of serum markers in patients with pancreatic cancer
- Authors:
- Published online on: March 29, 2020 https://doi.org/10.3892/ol.2020.11491
- Pages: 4106-4114
Abstract
Introduction
Pancreatic cancer (PC) is an aggressive malignant tumor with a low survival rate; the 5-year survival rate of this malignancy is <8%, making it the third major cause of cancer-associated mortality in the USA (1). It has been predicted that PC will become the second leading cause of cancer-associated mortality after lung carcinoma by 2030 (2). Such high mortality is caused by delayed diagnosis due to a lack of early diagnostic tools. Indeed, the early diagnosis of PC serves a vital role in disease prognosis and the design of personalized therapy. Due to the lack of overt symptoms at the early stage of PC, missed diagnosis rates range between 39 and 70% (3). Patients with advanced stages of PC demonstrate a more severe physical condition compared with those diagnosed in the early stage (4). In addition, certain tumor biomarkers play important roles in the early diagnosis, therapy and monitoring of PC, such as carbohydrate antigen (CA)19-9, CA242, vascular endothelial growth factor (VEGF), CA724 and CA125. However, although the levels of the aforementioned tumor biomarkers are elevated in certain individuals with PC, this is not the case for all, and so they have low levels of sensitivity and specificity (5). More strikingly, CA19-9 is a biomarker approved by the US Food and Drug Administration for PC diagnosis, with a sensitivity of 70–81%; however, it fails to detect PC in the early stages of disease (6). Therefore, new tumor biomarkers that serve significant roles in the early diagnosis of PC are urgently needed.
Currently, proteomics is considered a powerful tool for accurate monitoring and quantification of protein expression changes (7). Indeed, several proteomic technologies have been widely used for the identification of biomarkers, including two-dimensional electrophoresis, stable isotope labeling with amino acid in cell culture, two-dimension difference gel electrophoresis and isobaric tags for relative and absolute quantification (iTRAQ) (8). Previously, iTRAQ-based analysis has been used to quantitatively assess the changes in protein abundance in various biological samples, with high sensitivity and reproducibility (9). For instance, this approach has been successfully used to identify diagnostic markers of gastric and lung cancer (8,9).
The iTRAQ technology is increasingly used in the field of cancer research; plasma or serum protein analysis via quantitative proteomics of patients with PC reveals cancer-associated proteins and polypeptides in comparison with specimens from non-diseased and chronic pancreatitis controls (10). Thus, the present study aims to assess serum proteins in patients with PC and healthy controls, providing a basis for screening serum biomarkers, which would be used in the early diagnosis of PC.
Materials and methods
Subjects
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.
Table I.Number of patients with pancreatic cancer at each stage of the 8th edition of the American Joint Committee on Cancer staging system (12) for pancreatic cancer. |
Serum collection and preservation
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.
Depletion of highly and lowly abundant proteins
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.
Protein quantification and SDS-PAGE electrophoresis
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.
Peptide extraction and iTRAQ labeling
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.
Separation of iTRAQ-labeled peptides under high pH reversed phase (RP) conditions
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.
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.
Database analysis
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.
Gene ontology (GO) and Igenuity® Pathway Analysis (IPA) analyses of differentially expressed proteins
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.).
Statistical analysis
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.
Results
Characteristics of patients with PC
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.
Table III.Clinical data and carbohydrate antigen levels in healthy controls (male) and patients with pancreatic cancer (male) (mean ± SD) |
Qualitative and quantitative analyses of serum proteins following immuno-affinity depletion of highly abundant proteins
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.
GO analysis
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.
Table IV.Analysis of the 76 differentially expressed proteins identified in the serum of patients with pancreatic cancer and healthy controls. |
Pathways identified by IPA
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).
Discussion
CA199 is a tumor-associated antigen (13). It is present in the majority of healthy individuals. Not all individuals are able to produce similar levels of CA19-9; the levels are closely associated with physiological characteristics and genetic status (13). The physiological level of CA19-9 varies from individual to individual due to their different genotypes. In the serum of those with Lewis (Le) antigen Le-a (+) and Le-b (−) genotypes, CA19-9 is maintained at a certain level (14). The serum CA19-9 level of Le-a (−), Le-b (+) genotype was relatively low. CA19-9 could not be detected in the serum of individuals with Le-a (−), Le-b (−) genotype. In individuals who are negative for Lewis antigen, CA19-9 was not significantly elevated despite the PC tumor being large. Despite CA19-9 being a PC-specific antigen, its specificity and sensitivity to the PC tumor are low, which makes it difficult to use CA19-9 as a biomarker in screening for the early diagnosis of PC, although it is a common method (14). CA19-9 has been detected in various other types of cancer, including pancreatic (84%), gallbladder (69%), colorectal (39%) and ovarian (35%) cancer (15). Pancreatitis, hepatitis, biliary inflammation and obstructive diseases have been reported as the most common benign diseases exhibiting elevated levels of CA19-9 (15). The diagnostic sensitivity of CA19-9 for PC decreased when the critical value of CA19-9 increased and when the specificity level increased. When the critical value of CA19-9 reached 100 ku/l, the sensitivity and specificity were 68 and 98%, respectively (14). Even though CA19-9 is considered a good diagnostic marker for PC, it should not be used for screening of the early stages of PC, as the levels of CA19-9 are rarely elevated in this stage (14). Even when the PC lesions have been detected using CT, but the tumor size was <3 cm, only 50% of the patients had elevated CA19-9 (14). The most important role that CA19-9 serves in PC is to evaluate the recurrence of PC and the efficacy of radiotherapy and chemotherapy following surgery via assessing the changes in the levels of CA19-9. Despite the level of CA19-9 in patients with PC being increased >10 times in the present study, it could not be used as a direct indicator for the diagnosis of PC.
The present study assessed serum proteins in patients with PC and healthy controls and identified a total of 531 proteins using iTRAQ technology. Among them, 442 were quantitatively analyzed. Several differentially expressed proteins were observed in subjects with PC in comparison with healthy controls, including APAF-1, RAD50 and TGF-β1. GO and IPA analyses demonstrated that the differentially expressed proteins mainly participated in the extracellular matrix component, receptor function, enzyme regulation, nucleic acid binding transcription factor activity and metabolic-associated biological process. In addition, protein-protein interaction analysis using the IPA software also revealed marked alterations of APAF-1, RAD50 and TGF-β1 in patients with PC.
As presented in Fig. 5 and Table IV, APAF-1 was significantly downregulated by 10-fold in serum samples from patients with PC compared with the normal group. It has previously been demonstrated that APAF-1 is an essential and necessary component of apoptotic bodies in the intrinsic apoptotic pathway (16). APAF-1 serves an important role in the mitochondrial apoptotic pathway, primarily affecting the normal apoptosis of cells (17). Loss of heterozygosity and DNA methylation can inactivate APAF-1, which is a common phenomenon in numerous types of cancer in humans, including PC (18,19). It has also been reported that when APAF-1 is inhibited in the pancreatic duct, it can result in the development of PC (17).
As an important component of the Mre11-Rad50-Nbs1 complex (20), RAD50 is primarily involved in maintaining chromosome stability. In the present study, RAD50 was increased by 2.2-fold in serum samples from patients with PC compared with the normal group. A mutation analysis of 32 double-stranded DNA damage repair genes in breast cancer and PC revealed that RAD50 inactivation was more likely to promote the development of PC (21); however, high expression of RAD50 is conserved (22). It is not yet understood whether RAD50 protein upregulation in patients with PC is a protected molecular mechanism or whether the upregulation is maintained via negative feedback regulation; further research is required in order for the underlying mechanism to be elucidated.
TGF-β plays an important role in cell cycle arrest, apoptosis, homeostasis, wound healing and immune regulation. In the case of cancer, TGF-β plays a dual role in different situations, functioning as a tumor suppressor or oncogene in the early disease stages (23). TGF-β has three isoforms, including TGF-β1, TGF-β2 and TGF-β3; among them, TGF-β1 is the most abundant in humans. The TGF-β signaling pathway consists of several stages, starting with TGF-β1 activation and release, followed by binding to three high affinity receptors (24). TGF-β binds to TGF-βR1 and TGF-βR2 receptors on the cell surface, forming dimeric-SMADs following activation. A previous study demonstrated that TGF-βR2 is one of the 16 most commonly mutated genes in PC (25). In multiple types of human cancer, the TGF-β signaling pathway is activated (26). As presented in Table IV, TGF-β1 was 1.67-fold less expressed in serum samples from patients with PC compared with control values. Through IPA analysis of upstream regulatory genes of TGF-β1, it was also revealed that significantly downregulated TGF-β1 may be due to the suppression of hypoxia inducible factor 1 (HIF1) α. According to a previous report, HIF1α plays important roles in the growth, invasion and metastasis of PC (27). Significant changes in TGF-β1 expression indicate that TGF-β1 may be one of the diagnostic indices for PC.
The present study demonstrated APAF-1, RAD50 and TGF-β1 should be considered candidate biomarkers for PC diagnosis. However, due to the limited sample size, further studies with larger sample sizes are required in order to confirm the identified differentially expressed proteins and validate their values in the diagnosis of PC. The results of the present study could aid in identifying future therapeutic drugs for the treatment of PC.
Acknowledgements
Not applicable.
Funding
The present study was supported by The Scientific and Technological Project in Shanxi (grant no. 100311099-3).
Availability of data and materials
All data generated or analyzed during the present study are included in this published article.
Authors' contributions
KJ and XD conceived and designed the study. KJ and XZ performed the experiments and data analyses. KJ wrote the manuscript. KJ and XD helped to revise the manuscript. XZ contributed to discussions and provided reagents/materials/analysis tools important for the completion of this work.
Ethics approval and consent to participate
All procedures performed in the present study that involved human participants were in accordance with the ethical standards of the institutional and/or national Ethics Committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The present study was approved by the ethics committee of Shanxi Provincial Cancer Hospital (approval no. 201732). All the patients included in the present study provided written informed consent.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
PC |
pancreatic cancer |
iTRAQ |
isobaric tags for relative and absolute quantification |
GO |
gene ontology |
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