Chinese Chemical Letters  2017, Vol. 28 Issue (5): 1093-1098   PDF    
Discovery of lung squamous carcinoma biomarkers by profiling the plasma peptide with LC/MS/MS
Yu Liu, Xiao-Hong Xun, Jian-Ming Yi, Yang Xiang, Jie Hua     
College of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang 414006, China
Abstract: Biomarkers can be used for the screening and clinical diagnosis of cancer, and peptidomics approach has been proven successful in the research of biomarkers. To develop better peptidomic technologies for fast, accurate, and reliable detection of peptides biomarkers for lung cancer, we have improved the procedures of blood collection to minimize the degradation of the blood proteins and optimize the extraction of peptidome peptides from plasma samples based on acetonitrile precipitation associated with size exclusion chromatography (SEC). Studies show that squamous cell carcinomas are found to express CAGE1, SPAT9 and TEX28 genes at significantly higher rates, and the results suggest that as tumors progress, the level of CAGE1, SPAT9 and TEX28 genes are likely to increase and lead to immunization. This suggests a potentially important therapeutic method for cancer testis-based cancer vaccines.
Key words: Lung cancer     Lung squamous carcinoma     Biomarkers     Cancer testis antigen     Peptidomics    
1. Introduction

Lung cancer is the most important cause of cancer deaths globally, and it accounts for 25% of all cancer deaths [1], however, a great many clinical practices indicate that early detection of lung cancer is a promising approach to lowering its incidence and mortality rate, for it allows effective intervention to be made [2].

Lung cancer is classified into two major groups: small cell lung carcinoma (SCLC) and nonsmall cell lung carcinoma (NSCLC). Major histologic types of NSCLC include adenocarcinoma, squamous cell carcinoma (SCC), bronchoalveolar carcinoma (BAC), and large cell carcinoma [3]. Traditionally, abnormal chest imaging has led to the suspicion of lung cancer, and test will be done to confirm the diagnosis. Most frequently used measures include computerized tomography (CT), computerized axial tomography (CAT) scan, low-dose helical CT scan (or spiral CT scan), magnetic resonance imaging (MRI) scan, positron emission tomography (PET) scan, bone scans, and also sputum cytology, bronchoscopy, etc. [4].

Biochemical screening is anotherchoice.This isbased onthe facts that, astumorsdevelop, thecellsand/or the organs release numerous DNA, proteins, peptides and metabolites, and the amounts of these substances are associated with the stages of tumors, and therefore they can be used as biomarkers for screening and clinicaldiagnosis of cancer [5], and hundreds of biomarkers have been extensively investigated in the last several decades.Although biomarkers such as carcinoembryonic antigen (CEA), cytokeratin-19 fragment (CYFRA) [6], squamous cell carcinoma antigen (SCC), and neuron-specific enolase(NSE), CD59 glycoprotein, transthyretin (TTR), GM2 activator protein (GM2AP) [7] etc. have been found holding great promise for cancer detection, diagnosis, and prognosis, there is a need for further studies on their sensitivity and specificity. It should be pointed out that so far there is not a specific biomarkeridentified for lung cancer detection in clinical diagnosis and screening [8], and studies show that a panel of biomarkers are likely to be more effective for proper disease diagnosis [9, 10]. For example, Patz [39_TD$DIF]et al. have shown that by using a protein biomarker panel of CEA, retinol binding protein (RBP), R1-antitrypsin (AAT), and squamous cell carcinoma (SCC) antigen, they are able to classify 88% of patients with lung cancer and 82% of patients without cancer correctly [10].

We are now focusing on the investigation of a novel panel of biomarkers for lung cancer detection. As only trace levels of biomarkers exist in the early stages of lung cancer, it remains a great challenge to establish robust approaches for the detection of trace biomarkers. Moreover, there are various biomarkers which need to be detected with different technologies such as proteomics, peptidomics, and metabolomics. The term peptidomics is not introduced until the beginning of the year 2000 due to the fact that peptidomics analysis is only made possible after several advances in mass spectrometry (MS) and related techniques and progresses in genome projects that delivered comprehensive data pools for peptidomics studies [11-13]. Although the peptidomics approach has been proven successful in the research of biomarkers [14, 15], we are in need to develop better peptidomic technologies for fast, accurate, and reliable detection of peptides biomarkers in the early stages of the disease. Actually, it is a great challenge to determine whether the blood has detectable peptidome that can really reflect the changes due to the perturbations introduced by specific disease states, since studies show that most blood peptidome peptides reported are products from the degradation of common blood proteins [16-19]. This issue also reveals the importance of properly dealing with the procedures associated with sample collection, processing, storage, etc. In the present study, we try to improve the procedures of blood collection to minimize the degradation of the blood proteins and optimize the extraction of peptidome peptides from plasma samples based on acetonitrile precipitation associated with size exclusion chromatography (SEC), and provide these peptides to the label-free mass spectrometric quantification analysis combined with the statistical analyses on Expressionist proteome server platform.

2. Results and discussion 2.1. Enrichment peptidome from serum

Since reproducible and accurate separation of the peptidome fraction from serum was essential for the effective screening of biomarkers, we have optimized a simple gel filtration chromatography method and evaluated the peptide recovery. To avoid uncontrolled degradation of serum components arising from intact proteases and peptidases, all serum samples were collected using the PD100 column (BD, USA). Proteins are either precipitated with acetonitrile or subjected to thermal denaturation. The eluent is subjected to analyze by gel filtration high pressure liquid chromatography (Fig. 1).

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Figure 1. Gel filtration chromatography of serum samples (A: by thermal denaturation; B: precipitated with acetonitrile).

The results indicate that more proteins can be precipitated by mixing acetonitrile with serum when proper proportion is adopted. In addition, after thermal denaturation, the peak with retention time of 39.5 min in Fig. 1A is proven to be a small compound with molecular weight of 500 Da, which shows that proteins or peptides have degraded into smaller molecules in the process of degeneration. Such degradation is harmful for subsequent analysis. For the best effect of precipitation, we compared the effect of different proportions of acetonitrile precipitation. Then, the size exclusion chromatography is assessed by analyzing the fraction the retention time from 12-36 min with the MALDITOF mass spectrometer. In order to make clear analysis and comparison, the range 1900-2400 Da is selected for signal collection. The results of five precipitations caused by different proportions are shown in Fig. 2.

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Figure 2. MALDI-TOF mass spectrometer of the five precipitations (volume ratios of acetonitrile to plasma:A:1; B:1.5; C:2.0; D:2.5; E:3.0).

No peaks are found when one and 1.5 times volume of acetonitrile to plasma are adopted, while five peaks are found using 2 times and 3 times volume of acetonitrile to plasma, and the strongest signal of the peak is achieved when the ratio is 2.5:1, indicating the best precipitation degeneration. It also shows that this method is satisfactory and can be applied in the peptidomics analysis. Even though recent mass spectrometry instruments have allowed measurements of peptide mixtures at high sensitivity [20], enrichment of targeted peptides is still indispensable to achieving detection and identification of serum components in limited amounts of biological materials. In this case, the methodology to purify unabridged samples without loss of targeted components is crucial. The previous peptidome extraction technologies, such as ultrafiltration [21] and solid phase extraction [22], are covered limited, and magnetic beads extraction of peptidome is expensive [23]. However, our peptidome extraction technology consisting of acetonitrile precipitation and gel-filtration chromatography is simple and enables us to detect the peptides in the desired mass range, which shows its unique advantages in the application of peptidomics analysis.

2.2. Peptide biomarker screening for lung squamous carcinoma

To explore serum peptides which could be applied for early detection of lung squamous carcinoma, we acquired quantitative peptidome profiles from 70 lung squamous carcinoma patients that consistof12stage-Ipatients, 14stage-IIpatients, 15stage-IIIapatients, 16 stage-IIIb patients, 13 stage-Ⅳ patients to identifycandidate serum biomarkers for lung squamous carcinoma. The serum samples are purified using gel filtration chromatography as described above and individually subjected to LC/MS/MS analyses using API3200 mass spectrometry. The peptide sequencing is performed by a combination of LC/MS/MS analysis and MASCOT database search. 80 candidate peptides are identified from lung squamous carcinoma compared to normal control (Table S1 in Supporting information). Regarding the 80 candidate peptides, 9 peptides are uniquely identified (Table 1, Fig. S1 in Supporting information).

Table 1
Target peptides identified from the 80 candidate peptides.

Three of them are fragments derived from fibrinopeptide A (FPA) which is N terminally cleaved product from fibrinogen A (FIBA). In fact, the results suggest that FPA fragments are potential lung cancer-associated biomarkers showing the significant increase of concentrations in lung cancer patients's sera. However, since anomalous turnover of FPA was previously reported in several other diseases including gastric cancer [24], diabetic nephropathy [25], coronary heart disease [26], these FPA fragments could not be defined as lung squamous carcinoma specific biomarkers [27]. 1433z belongs to high conserved protein family which is ubiquitously expressed in mammalian cells. 1433z can form a dimmer which interacts with a variety of proteins including protein kinase, receptor, and other signaling proteins. 1433z protein plays a role in suppression of apoptosis [28]. Overexpression of 1433z protein is frequent event in many tumors such as breast cancer, gliomas, lung cancer and so on. 1433z might be used as a novel molecular marker in prognosis [29]. K1C17 is expressed in the outer root sheath and medulla region of hair follicle specifically from eyebrow and beard, digital pulp, nail matrix and nail bed epithelium, mucosal stratified squamous epithelia and in basal cells of oral epithelium, palmoplantar epidermis and sweat and mammary glands [30]. Overexpression of K1C17 promotes epithelial proliferation and tumor growth by polarizing the immune response [31]. K1C17 has been already identified as a biomarker for lung squamous carcinomas, but not identified in lung adenocarcinoma. K1C17 protein is a useful marker in differential diagnosis of lung squamous carcinomas and lung adenocarcinoma [32, 33]. Haptoglobin in general, as well as the differentially glycosylated forms of this abundant plasma protein, has the strongest clinical evidence as a general cancer biomarker [34, 35]. In lung cancer, the levels of serum haptoglobin and its different glycoforms have been demonstrated as potential biomarkers for both small cell lung cancer [36] and NSCLC [37]. Complement C9 as a cancer biomarker in plasma was not reported except Guergova-Kuras et al. [38]. Guergova-Kuras et al. thus provided the first independent validation of this biomarker using a quantitative immunoassay on two independent clinical cohorts with 264 lung cancer cases and 232 healthy controls. The C9 component of the complement system has not been associated as a plasma marker with cancers of any type [38].

CAGE1 is a testis-specific expression gene in normal tissue, but widely expressed in some cancer tissue and cell lines. Diseases associated with CAGE1 include carotid artery occlusion and lung squamous cell carcinoma [39]. Liu et al. demonstrated that MASPIN, which is also known as Serpin B5, is a novel tumor suppressor. CAGE1 genes are identified as being uniquely expressed when MASPIN is knocked down in cell lines, which are associated with the development and progression of tumors [40]. NYD-SP16 contains 1595 base pairs (bp) and a 762-bp open reading frame encoding a 254-amino acid protein with 73% amino acid sequence identity with the mouse testis homologous protein. Multiple tissue distribution indicates that NYD-SP16 mRNA is highly expressed in the testes and pancreas, with little or no expression elsewhere. NYD-SP16 expression is 6.44-fold higher in adult testis than in fetal testis. NYD-SP16 protein may play an important role in testicular development/spermatogenesis and may be an important factor in male infertility [41]. TEX28 is expressed in testis. Ocular gene expression experiments conducted revealed the presence of TEX28 in five ocular tissues, including the retina and the sclera at the mRNA level [42]. Few studies on TEX28 have been published. The function of the TEX28 is known very little [43].

2.3. Cancer-testis proteins in lung squamous carcinomas

A total of 60 cases of lung squamous carcinomas are typed for cancer-testis proteins expression. The cancer-testis proteins expression is estimated by non-competitive ELISA. Among the lung squamous carcinomas tested, 41 (68.3%) expressed at least one of the three cancer-testis proteins tested. The most frequently observed cancer-testis protein is TEX28, presenting in 46.7% of the samples, followed by CAGE1 (26.7%) and SPAT9 (11.7%) (Table 2, refer to Table S2 in Supporting information). Cancer-testis proteins form a large family of protein coding sequences with specific expression pattern: They are expressed in the testis and certain types of cancers [44, 45]. In the present study, we did not observe the expression of the three cancer-testis proteins in normal control blood plasma.

Table 2
Cancer-testis proteins expression estimated by non-competitive ELISA in peptidome samples of 60 cases of lung squamous carcinomas.

Cancer-testis proteins are predominantly expressed in normal gametogenic tissues as well as in different histological types of tumors [46, 47]. In testis, cancer-testis proteins are expressed exclusively in cells of the germ cell lineage, although there is a marked variation in the protein expression pattern during different stages of sperm development. Likewise, a heterogeneous expression is observed in tumors [48, 49]. Because of their restricted expression pattern, cancer-testis proteins are considered ideal targets for cancer immunotherapy [48]. Indeed, a small subset of patients immunized with the known cancer-testis proteins MAGEA and NY-ESO-1 have shown clinical benefits after immunization [49, 50]. However, as cancer-testis proteins are expressed in only a small subset of human tumors and in only afraction of cases of a given tumor type, the identification of additional cancer-testis proteins is crucial for improving current immunotherapy protocols.

3. Conclusion

In conclusion, our approach of acetonitrile precipitation and size exclusion chromatography are suited to prepare the plasma proteome from complex plasma samples. This technology provides a rapid way to prepare large numbers of candidate peptides in complex samples. Our approach of screening the plasma proteome with LC/MS/MS not only delivered the results consistent with previously reported lung cancer biomarkers but also uncovered cancer testis proteins. In this study, squamous cell carcinomas are found to express CAGE1, SPAT9 and TEX28 genes at significantly higher rates. Most cancer testis antigens are immunogenic, and their use as therapeutic cancer vaccines have been systematically evaluated [49-52]. Our results suggest that as tumors progress, the level of CAGE1, SPAT9 and TEX28 genes they express are likely to increase and lead to immunization. This also suggests a potentially important therapeutic method for cancer testis-based cancer vaccines.

4. Experimental 4.1. Human plasma samples and collection procedure

Human blood plasma samples are collected with informed consent from the patients who are suffering from the lung squamous carcinoma (stage Ⅰ-Ⅳ) at the Second Hospital of Yueyang. Human plasma samples as normal controls are also obtained with informed consent from 35 healthy volunteers who received medical checkup at the Second Hospital of Yueyang. To circumvent undesirable degradation of proteins and peptides, the same strict laboratory protocol is applied for the treatment of each sample. Briefly, all venous blood specimens are collected with vacuum blood collection tubes BD P100 Kit (Becton Dichinson Company, NJ USA). After collecting the blood, plasma fractions are separated with centrifugation at 1500 × g for 15 min at 4 ℃ and immediately stored at -80 ℃. One freeze-and -thaw procedure is permitted for any plasma samples used in the present study. This study is approved by the Ethical Committee of the Second Hospital of Yueyang, and the Ethical Committee of Hunan Institute of Science and Technology.

4.2. Acetonitrile precipitation of plasma samples and subsequent peptidome enrichment

All plasma samples are frozen and thawed once and immediately incubated at room temperature for 30 min after mixed with 2.5 times acetonitrile. The mixed samples are separated with centrifugation at 10, 000 × g for 30 min at 4 ℃. After filtration with Spin-X 0.45 μm spin filters (Corning Incorporated, Corning, NY, USA), the samples are loaded into a Zorbax psm 60 column (6.2 × 250 mm, Agilent, USA) coupled with 1525 HPLC system (Waters, USA). Each peptidome fraction is collected from 12 to 36 min in the constant flow of 5% acetonitrile at 0.5 mL/min flow rate. The collected fractions are dried-up with vacuum freeze drying (Scientz Corporation, Ningbo, China).

4.3. LC-MS/MS experiments

HPLC is performed on a 1.0 mm × 100 mm i.d. LC column containing C4-bonded particles (3-μm, 300 Å size, Thermo Scientific Dionex, USA) using LC-20AD system (SHIMADZU, Japan). The peptidome sample is injected into the LC column, and separated with a gradient generated by replacing mobile phase A (acetonitrile/H2O/formic acid, 2:98:0.1, v/v/v) and B (acetonitrile/H2O/formic acid, 70:30:0.1, v/v/v) with the multistep linear gradient of solvent B 5 to 60% for 20 min and 60% for 10 min and 60%-95% for 10 min at a flow rate 60 μL/min. The peptidome samples are analyzed by triple quadrupole LC/MS/MS (AB SCIEX, USA). The parameters on ABI3200 are shown as follows: CUR: 10; IS: 5500; TEM: 300; GS1: 50; GS2: 50; ihe: ON; CAD: 6; DP: 50; EP: 80; CE: 35; CXP: 8. The MASCOT database search is performed on the Analyst QS 2.0 software (AB Sciex, Foster City, CA, USA). The MS/MS data is searched against the human protein database from SwissProt 57.4 (20, 400 sequences) using the search parameters: taxonomy = Homo sapiens, enzyme = none, fixed modifications = none, variable modifications = oxidation (Met), MS tolerance = 50 ppm, and MS/MS tolerance = 0.1 Da, with mascot automatic decoy search. We use the homology threshold for less stringent criteria providing almost same protein identification numbers with the criteria expectation value < 0.05.

4.4. Cancer testis peptide synthesis and purification

The peptides are synthesized starting from a PAL-PEG-PS resin on an automatic peptide synthesizer (PerSeptive Biosystems, America) using an Fmoc/tert-butyl strategy and HOBt/TBTU/NMM coupling method. Peptide synthesis is accomplished on a 0.1-mmol scale. The terminal Fmoc group is removed by treatment with 1:4 piperidine/ N, N-dimethylformamide (v/v). After completion of synthesis, the peptide is cleaved from the resin with simultaneous removal of side chain protective groups by treatment with reagent K (82.5% trifluoroacetic acid, 5% double distilled H2O, 5% phenol, 5% thioanisole, and 2.5% ethanedithiol) for 2 h at room temperature. The resin is then filtered, and the free peptide is precipitated in cold ether at 4 ℃. After centrifugation and washing once with cold ether, the peptide is dissolved in 20% acetic acid and lyophilized. The reduced peptides are purified by semipreparative reverse-phase HPLC (Waters 1525 HPLC, Waters Corporation, Milford) using a 45min linear gradient of 5%-50% eluent B (0.1% trifluoroacetic acid in acetonitrile) in eluent A (0.1% trifluoroacetic acid in double distilled H2O) over 45min on a C18 column (Luna, 10 ×250mm) at 2mL/min flow rate. Fractions are analyzed by analytical HPLC, and more than 95% pure fractions are pooled and lyophilized. The molecular weights of the peptides are checked by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry(UltraFlexI, BrukerDaltonics).Forpeptides, themeasured molecular mass corresponds to the predicted value within 1.0 unit, which is consistent with the correctness of the sequence and the complete removal of all side chain protection groups.

4.5. Cancer testis protein ELISA test

Blood plasma samples from the patients are taken to check SPAT9, CAGE1 and TEX28 antibody. Non-competitive enzyme immunochemistry method is used to determine the cancer testis protein antibody. The peptide is dissolved in coating buffer and diluted to 2 μg/mL.ThesolutionSPAT9-1ismixedwithSPAT9-2(orCAGE1-1is mixed with CAGE1-2; or TEX28-1 is mixed with TEX28-2). 200 μL peptides mixture solution is then inoculated to the each well of the ELISA plate and incubated at 4 ℃ for 24h. After washing the well three times with PBST (10mmol/L PBS (pH 7.00) containing 0.05% Tween 20), the peptide antigen-coated plate is incubated with 200 μL of PBS (10mmol/L phosphate buffered saline, pH 7.0, containing 0.05v/v% Tween 20 and 5% BSA) at room temperature for 30min. The plate is then washed 4 times with 200 μL of PBST. 50μL of plasma sample is then inoculated and incubated at room temperature for one hour. After washing the wells four times with 200 μL of PBST, 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-human IgG is added to each well and incubated at room temperature for one hour. After washing the wells four times with 200 μL of PBST, 100 μL of tetramethylbenzidine (TMB) solution is used to develop color reaction (10min) according to manufacturer's instruction.The opticaldensity(OD)of the solution is determined by a microplate reader at 450nm after terminating the reaction by adding 100 μL of 2mol/L sulfuric acid.

4.6. Statistical analysis

Results were presented as mean ±SEM. Statistical analysis was carried out by Student's t-test, or a one-way or two-wayanalysis of variance followed by Student-Newman-Keuls' or Bonferroni posttests when appropriate. P < 0.05 were considered to be statistically significant.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 21172067), Science and Technology Project of Hunan Province (Nos. 2013SK2015, 2014SK3017), Scientific Research Key Fund of Hunan Provincal Education Department (No. 13A032).

Appendix A. Supplementary data

Supplementary data associatedwith this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.11.026.

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