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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 1  |  Page : 96-103

Isobaric tags for relative and absolute quantitation-based proteome analysis of Vietnamese colorectal carcinoma tissues


Department of Biotechnology, School of Biotechnology, International University, Vietnam National University HCMC, Quarter 6, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam

Date of Web Publication13-Mar-2019

Correspondence Address:
Dr. Thi Thu Hoai Nguyen
School of Biotechnology, International University, Vietnam National University HCMC, Quarter 6, Linh Trung Ward, Thu Duc District, Ho Chi Minh City
Vietnam
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.202889

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 > Abstract 


Context: Colorectal cancer (CRC) is one of the most common malignancies and one of the leading causes of cancer death worldwide. Establishing early detection methods or markers of CRC is central to improve the survival rate of CRC patients. Nowadays, new molecular tools have been developed to acquire further knowledge on tumor progression.
Aims: Comparative proteomics analysis of Vietnamese colorectal carcinoma in different stages was performed to gain an insight into the molecular events taking place in CRC and metastasis.
Subjects and Methods: In this study, the comparative protein expression analysis of ten paired CRC and its corresponding noncancerous tissue samples was performed using the combination of isobaric tags for relative and absolute quantitation labeling and mass spectrometry (MS). The data obtained were further analyzed with Ingenuity Pathways Analysis (IPA) system.
Results: Based on the MS/MS spectra analyzed by ProteinPilot software, 684 proteins were identified, out of which 215 were observed to be differentially expressed in at least 1 sample pair. Individual protein expression and variation have been identified for each patient. IPA system demonstrated cytoskeletal signaling as the top-ranked functional pathway network associated with the oncogenic function.
Conclusions: Our study supplemented the understanding about proteome of Vietnamese CRC patients and identified statistically protein expression differences among samples assisting in finding effective biomarkers for CRC diagnostics.

Keywords: Colorectal cancer, isobaric tags for relative and absolute quantitation, proteomics


How to cite this article:
Pham TT, Le KP, Vo PU, Le KM, Lim TK, Lin Q, Nguyen TT. Isobaric tags for relative and absolute quantitation-based proteome analysis of Vietnamese colorectal carcinoma tissues. J Can Res Ther 2019;15:96-103

How to cite this URL:
Pham TT, Le KP, Vo PU, Le KM, Lim TK, Lin Q, Nguyen TT. Isobaric tags for relative and absolute quantitation-based proteome analysis of Vietnamese colorectal carcinoma tissues. J Can Res Ther [serial online] 2019 [cited 2019 Oct 18];15:96-103. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/96/202889




 > Introduction Top


Colorectal cancer (also called colon cancer-CRC) is cancer that occurs in the colon or rectum. CRC affects men and women of all racial and ethnic groups with the variation in distribution,[1] and it is commonly found in people of 50 years old and older. In the United States, it is the third most often diagnosed cancer and the third leading cause of mortality due to cancer in both men and women with the estimated diagnosed cases of 136,830 and 50,310 deaths from the disease in 2014.[2] In Vietnam, data presented by GLOCOBAN in 2012 stated that CRC is the fifth common cancer with 8768 new cases diagnosed annually which contributed to 7.0% of all kinds of cancer.[3]

According to the American Cancer Society, CRC has been identified as a major priority because the application of existing knowledge about cancer screening and prevention has great potential to prevent cancer, reduce suffering, and save lives.[2] However, CRC is usually not detected at early stages where treatment has higher success rate due to the lack of sensitive and specific screening methods. Besides, the early signs of CRC are often equivocal and can be confused with other diseases such as irritable bowel syndrome, Crohn's disease, or peptic ulcers. This means that a lot of patients are diagnosed lately when cancer has already spread (metastasized) to other parts of the body.

Nowadays, proteomics - a field focused on the variations within proteome of a given biosystem and understanding the relationships therein has been developed rapidly and used widely in cancer research. In this field, modern tools and technologies such as two-dimensional (2D) gel electrophoresis, mass spectroscopy, or protein microarray are applied to extract important biological information which aid scientists and clinicians in understanding the association between protein alterations and malignancy as well as the effect of molecular intracellular mislocalization in tumor initiation.[4] With these efforts, effective diagnostic methods are expected to be improved by either discovering new biomarkers or serological tests that can be used for early detection of cancer, design individual therapies, and to identify underlying processes or mechanisms involved in the disease. Because of some limitations of gel-based methods such as low sensitivity and excessive time/labor cost, isotope-based quantitative proteomics such as isobaric tags for relative and absolute quantitation (iTRAQ) which allows multiplexing of up to eight samples to identify the relative abundance of proteins in different samples within a single liquid chromatography-mass spectrometry (LC-MS) analysis, have made breakthroughs in various fields of research. Applying this technique, several previous studies have found out promised proteins biomarker for the prognosis/diagnosis of CRC.[5],[6],[7] However, this high-throughput technique has not been applied in CRC research in Vietnam because of high cost of limitation in materials and tools.

This study was aimed to determine the differences in protein expression between CRC and normal tissues and establish the protein expression profile using 8-plex iTRAQ analysis. Our results can provide an insight into the molecular events of CRC metastasis as well as illustrating the power of iTRAQ quantitative proteomics approach in identifying important protein expression patterns of CRC metastasis. The finding from this study can be used as theoretical basis for further searching of the potential biomarkers and therapeutic targets relevant to human CRC occurrence and development.


 > Subjects and Methods Top


Proteomic samples preparation

Ten matched sets of CRC tissue samples and the paired adjacent normal mucosal ones were collected from hospitalized surgical patients after operation and transported on ice to our laboratory then proceeded immediately. The profiles of patients and clinical features of carcinoma tissues including age, gender, and tumor-node-metastasis stage were also recorded [Appendix 1].



To extract the total protein of the samples, each tissue sample which about 1 mm3 in sizes were washed at least twice with phosphate-buffered saline (PBS) containing 100 μg/mL ampicillin, submerged overnight at −20°C in rehydration buffer containing chaps, thiourea, and urea. Afterward, the ice-cold samples were sonicated on ice, centrifuged at 10,000 rpm in 10 min (4°C) to collect the supernatant.

Before proceeding further steps, all of protein samples were cleaned using 2D-Clean-Up Kit (GE Healthcare, USA). Protein concentration from each cell lysates was estimated by performing a Bradford protein assay.

Isobaric tags for relative and absolute quantitation labeling

To investigate the protein identification and expression variation among samples, an aliquot equivalent to 100 μg of each sample was removed for iTRAQ labeling.

Briefly, the protein which was dissolved in triethylammonium bicarbonate buffer was reduced with 1 μL tris-(2-carboxyethyl) phosphine, alkylated with 2 μL methyl methanethiosulfonate, and incubated at 60°C for 1 h. Then, 1 μL of Cysteine-Blocking Reagent was added to each sample tube and incubate at room temperature for 10 min before digesting with 10 μL trypsin for each tube at 37°C overnight.

The digested peptides were then labeled with respective isobaric tags in iTRAQ Reagent 8-Plex Kit (AB Sciex, USA), incubated at room temperature for 2 h before being combined. The iTRAQ workflow was based on the manufacturer's standard protocol. All the samples were divided into three sets of iTRAQ experiment as followed: iTRAQ Set 1 including sample pair 1, 2, 3, and 4; iTRAQ Set 2 including sample pair 1, 5, 6, and 7; iTRAQ Set 3 including sample pair 1, 8, 9, and 10. Each sample in an iTRAQ set was labeled with different iTRAQ tag (113–119 and 121, respectively) and then all the contents of each iTRAQ reagent-labeled sample tube in 1 set were combined into 1 fresh tube. The sample pair 1 was run in all three iTRAQ experiments as a reference sample.

Strong cation exchange chromatography

Strong cation exchange (SCX) chromatography was performed to remove all interfering substances including dissolution buffer, organic solvent such as ethanol, sodium dodecyl sulfate (SDS), calcium chloride, and excess iTRAQ reagents (Ghosh et al., 2011), iTRAQ-labeled mixture was purified and preseparated by SCX column. The experiment was strictly followed the manufacturer's instruction (AB Sciex, Singapore). The eluted fractions were desalted using a Sep-Pak® C18 cartridge and evaporated to dryness in a SpeedVac concentrator.

Two-dimensional liquid chromatography-mass spectrometry/mass spectrometry analysis

2D LC was performed using the Ultimate 3000 LC system (Dionex-LC Packings, Sunnyvale, CA, USA) to separate mixed peptides and then MS and MS/MS analyses were performed using the 5800 MALDI-TOF/TOF Analyzers (Applied Biosystems/AB Sciex). Mascot database search engine was employed, and Swiss-Prot database was selected searching for human sequence.

Data analysis

The raw MS/MS data were analyzed using ProteinPilot software version 4.5 (AB Sciex) for the protein identification and relative quantification. The proteins were identified by searching Swiss-Prot-UniProt protein database. For iTRAQ quantitation, the peptide for quantification was automatically selected by Pro Group algorithm to calculate the reporter peak area, error factor, and P value.

To optimize the number of identified proteins, a filter with strict cutoff criteria was applied including unused ProtScore >1.3 (a threshold of confidence above 95%) and a minimum of two distinct peptides identified for each protein. The resulting dataset was auto bias - corrected to eliminate any variations caused by the unequal mixing during the combination of differently labeled samples. The results were then exported into Microsoft Excel for manual data interpretation. The differentially expressed proteins were analyzed according to the gene ontology annotation using Ingenuity Pathways Analysis (IPA) for their molecular function, biological process, and cellular component.

Western-blot analysis of Vimentin

Ten micrograms of each protein sample extracted from tissue was separated by one-dimensional SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. After blocking the blots using 5% (w/v) bovine serum albumin (BSA) in PBS with 0.1% Tween 20 (PBS/T) for 1 h at room temperature, the membranes were incubated with primary antibody (rabbit anti-Vimentin antibody, Abcam) diluted 1:5000 in PBS-T contained 5% (w/v) BSA for 2 h. After that, the membranes were washed with PBS/T and incubated with secondary antibody (horseradish peroxidase [HRP]-conjugated goat anti-rabbit IgM, Abcam) diluted 1:5000 in PBS-T contained 5% (w/v) BSA for 2 h. Blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) and scanned within the linear range of detection by chemiluminescence Genesys Box Scanner (Syngene, USA). The Western-blot image was then captured, and the intensity of bands was quantified and compared using ImageJ software (Wayne Rasband, National Institutes of Health, Maryland, USA).


 > Results Top


Isobaric tags for relative and absolute quantitation proteomics profile of colorectal cancer samples

Three protein summary which contains identifications and quantitative data for three iTRAQ runs were exported into Excel files and then combined for comparison. First, following strict cutoff criteria were applied to filter out the qualified proteins: (1) Proteins with at least two identified peptides and (2) proteins with unused protein score ≥1.3 which corresponds to the 5% false discovery rate. Within an iTRAQ run, differentially expressed proteins can be determined based on the protein expression ratios between the normal and its paired-cancer sample from the same patient and P values provided by ProteinPilot. Proteins are considered differentially expressed if (1) the iTRAQ ratios are either >1.5 for upregulation or <0.667 for downregulation which corresponds to 1.5-fold-change and (2) P < 0.05.

In total, there were about 400–500 unique proteins identified by ProteinPilot software for each iTRAQ experiment (393, 426, and 571 proteins in iTRAQ 1, 2, and 3, respectively). Combining the results of three iTRAQ experiments, 684 unique proteins were finally identified with quantification information. Among them, 265 (38.6%) were detected in each of the three iTRAQ experiments, and 371 (54%) were common in at least two runs. Among 684 proteins identified by ProteinPilot software, 215 proteins were differentially expressed in at least one paired-sample. The individual protein expression of each patient is summarized in [Table 1].
Table 1: Protein expression summary of ten sample pairs

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Sample pair one was run separately in all three iTRAQ sets as internal control. As can be seen from [Table 1], the number of differentially expressed proteins are significantly higher in samples at mid- metastasis stage with ~70 proteins found to be changed in expression than at no-metastasis stage with about 35–60 differentially expressed protein. Particularly, the sample 9 which include the sample of CRC at late-metastasis stage had the most number of differentially expressed proteins.

Gene ontology

To further understand the molecular pathways associated with CRC development, a biological network analysis using IPA was performed with identified protein profiles. Among 215 proteins identified to be significantly altered in expression, 50% of them from the cytoplasm, 15% from the nucleus, 8% from the plasma membrane, and 27% from the extracellular space [Figure 1]. These proteins were also classified as their molecular functions by IPA [Figure 2].
Figure 1: Cellular distribution of all the differentially expressed proteins identified in three isobaric tags for relative and absolute quantitation sets

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Figure 2: Classification of significantly regulated proteins as per their molecular functions using ingenuity pathway analysis

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Using the IPA tool, the canonical pathways at different stages were analyzed, and the top 17 pathways were shown in [Figure 3]. The top-ranked pathways were pathways related to the adherence and movement (actin cytoskeleton signaling, epithelial adherence junction, integrin signaling…) or inflammation and immunity (acute phase response signaling, agranulocyte adhesion, and diapedesis).
Figure 3: Seventeen top most related canonical pathways in ingenuity pathway analysis

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Relevance of isobaric tags for relative and absolute quantitation dataset proteins to the process of colorectal cancer metastasis

[Table 2] summarized the expression profile of some interesting proteins in this study. We have observed a significant change in expression level of proteins involved in or associated with cytoskeletal signaling pathway including actin-binding proteins (caldesmon, calponin-1, filamin-A, gelsolin, synaptopodin-2, and transgelin) or intermediate filament proteins (desmin, synemin, and vimentin). Most of these proteins were found to be downregulated in this research except for vimentin. The expression pattern of vimentin was validated by Western blot.
Table 2: Differentially expressed proteins in colorectal cancer samples

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In addition, alpha-crystallin B chain (CRYAB), calreticulin (CRT), creatine kinase B level, heat shock protein beta-1 (HSPB1), and protein disulfide isomerase (PDI) are also differentially expressed proteins identified in our research and may play roles in metastasis progress.

Western blot

Vimentin was chosen to carry out Western blot to make a comparison between iTRAQ data and Western blot. According to our dataset, vimentin was considered to be upregulated in five CRC samples including sample 2, 5, 6, 7, and 9. As can be seen from Western-blot image [Figure 4] and the quantitating result of visual bands by ImageJ software [Table 3], the expression of vimentin was increased in CRC sample 2, 7, and 9.
Figure 4: Western-blot analysis of Vimentin. Ten micrograms of each protein sample 1–10; N: Normal tissue; C: Cancer tissue was used. Rabbit anti-Vimentin antibody (Abcam), HRP-conjugated goat anti-rabbit IgM (Abcam) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) were applied

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Table 3: Quantification result of Vimentin band by using ImageJ (Wayne Rasband, National Institutes for Health)

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 > Discussion Top


Metastasis is a critical event for cancer development with the involving of many processes including cell adhesion, migration, invasion, or proliferation. The significant changes on these pathways could lead to the biofunctional changes and relevant to the pathophysiology of CRC metastasis.

Interestingly, our iTRAQ data showed downregulation in most of the proteins associated with cytoskeletal signaling pathway. Cytoskeletal molecules involved in the regulation of cellular motility, adhesiveness, and other dynamic cellular functions so that it is to be expected that the altered organization of the actin cytoskeleton, which are interlinked to maintain a functional cell–cell system in epithelia, may induce events related to colorectal progressions such as cell migration, proliferation, and invasiveness.[8] Therefore, it is believed that the alteration of molecules relating to the actin cytoskeleton might become potential markers for the diagnosis of malignant tumors.[9]

Among this group of proteins, most differentially expressed proteins are actin-binding or actin-associated proteins such as caldesmon, calponin-1, filamin-A, gelsolin, synaptopodin-2, and transgelin. They were downregulated in 7–8 CRC samples among 10 samples investigated [Table 2]. These results were in agreement with previous studies, in which the reduced expression of these proteins in CRC was also observed.[10],[11],[12] Actin-binding proteins are multifunctional proteins which involved in the regulation of actin polymerization and the dynamic remodeling of the actin cytoskeleton. Therefore, they are thought to play roles in cell migration, adhesion, division, and development, which are essential factors in tumor invasive, development, and metastasis.[10],[13],[14],[15],[16] Particularly, some proteins in this group have been studied and developed as a biomarker for CRC diagnostic tests such as gelsolin and transgelin. Gelsolin is a 90-kD protein that involved in the regulation of actin polymerization by severing and capping actin filaments.[12] The research of Fan et al. stated that decreased expression of gelsolin can serve as a potential biomarker of colorectal adenocarcinoma.[17] Transgelin is another actin-binding protein that promotes cell motility and emerged as a top-ranked biomarker for CRC in recent years. However, while previous studies demonstrated the overexpression of transgelin in CRC samples,[18],[19] our study showed the opposite result, in which transgelin expressed with low level in most of cancerous tissues.

Beside actin-associated proteins, our study also identified the downregulation of several intermediate filament-associated proteins in CRC cells including desmin and synemin and the other cytoskeletal signaling-related proteins such as filamin-C, talin, myosin, and tropomyosin family members. Especially, vimentin – a Type III intermediate filament protein which plays a role in regulating epithelial–mesenchymal transition[20] was presented to be upregulated in 5 out of 10 CRC samples in our study. This protein was also observed with increased expression and identified as potential predictive biomarkers for identifying CRC patients in research of Toiyama et al.[20]

Our data also showed that colorectal adenocarcinomas possess lower expression level of chaperones and heat shock proteins such as HSPB1 and CRYAB than the corresponding normal tissues. These proteins have been known to play critical roles in modifying the structures and interactions of other proteins as well as in defense against cellular stress and apoptosis.[21],[22] Therefore, their downregulation might contribute to the development of human cancers including CRC.

Furthermore, another noteworthy protein which found to be overexpressed in 6 out of 10 CRC samples in this study is CRT. CRT is normally found in the lumen of the endoplasmic reticulum (ER) with two major functions: protein chaperoning and regulation of Ca2+ homeostasis.[23] While some of the reports indicated that CRT expressed much more strongly in CRC tumor tissues compared to normal tissue,[24],[25] the other ones showed the downregulation of CRT in CRC sample.[26],[27] Besides, as be discussed in several studies, CRT involved in the regulation of cell adhesion which is an essential process of cancer metastasis through direct interaction with the integrins α-subunits.[28],[29],[30] Furthermore, many studies have stated that alteration of CRT levels had effects on tumor cell proliferation and non-ER CRT also regulates other important biological functions such as gene expression and RNA stability.[31],[32]

In addition, decreased expression levels of creatine kinase B level - an enzyme involved in energy transduction pathways[33] and overexpression of PDI - an enzyme that catalyzes disulfide formation and isomerization and a chaperone that inhibits aggregation[34] was observed in several metastatic samples [Table 2].

In recent years, many researches have been carried out to evaluate the alteration in the global protein landscape of CRC. However, only a few proteins have been evaluated as potential biomarkers for CRC but with low specificity and sensitivity such as carcinoembryonic antigen (CEA) or carbohydrate antigen 19-9.[35],[36],[37] However, in this study, the level of CEA has been identified with no significant difference between cancerous tissues and their corresponding normal ones.

There are several studies which also applied iTRAQ quantitative proteomics technique to explore the molecular mechanism of CRC metastasis[5],[6] but with several differences in experimental design with our study and variation in result. For example, the project of Ghosh et al. used protein extracted from two primary and metastatic CRC cell lines instead of from patient tissues and it has been also found that significantly differentially expressed proteins identified in their experiments were associated with cytoskeletal signaling, cellular adhesion, migration, or cell death.[6] Besides, there are other studies, in which their findings were in contrast with our ones including the upregulation of actin-related proteins[7] or HSPB1.[38]


 > Conclusions Top


In summary, the obtained data can be used to improve the understanding about pathophysiology of CRC metastasis as well as serve as the key information for further study with the expectation of identifying an effective molecular target for diagnostic or therapy of this disease. Our study found several proteins which presented their change in expression even at early-metastasis stage such as calponin-1, CRT, synaptopodin-2, or transgelin and could be a promising indicator for CRC prediction.

Acknowledgment

We would like to give special thanks to Cho Ray Hospital and Dr. Tran Minh Thong, Department of Anatomic Pathology, Cho Ray Hospital, Ho Chi Minh City, Vietnam. Without their support, we could not initiate the study. We thank Vietnam National University of HCMC, for the finance support to this study (C2014-28-05). We also thank the National University of Singapore and Dr. Russell Ronald Braeuer, for supporting us technical issues.

Financial support and sponsorship

This study was financially supported by Vietnam National University of Ho Chi Minh City, Vietnam.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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