- Email: [email protected]
In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy
Accepted Manuscript In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy Aihua Hou, Kai Pong Law, Min Qi Tin, Yoon Pin Lim, Louis Tong PII:
S0014-4835(16)30323-2
DOI:
10.1016/j.exer.2016.10.006
Reference:
YEXER 7034
To appear in:
Experimental Eye Research
Received Date: 9 May 2016 Revised Date:
26 September 2016
Accepted Date: 6 October 2016
Please cite this article as: Hou, A., Law, K.P., Tin, M.Q., Lim, Y.P., Tong, L., In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy, Experimental Eye Research (2016), doi: 10.1016/j.exer.2016.10.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In vitro Secretomics study of Pterygium-derived Fibroblasts by iTRAQ-
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based Quantitative Proteomics Strategy
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3 Aihua Hou 1,a, Kai Pong Law 2,a, §, Min Qi Tin 1, Yoon Pin Lim 2,5 and Louis Tong 1,3,4,5*
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Ocular Surface Research Group, Singapore Eye Research Institute, Singapore
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Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore
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Duke-NUS Graduate Medical School, Singapore
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Singapore National Eye Center, Singapore
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Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore
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Equally contributed authors
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* Correspondence authors:
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Address: 20 College Road, The Academia, Level 6, Discovery Tower Singapore 169856, Tel: +65-
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65767213; Fax: +65- 62252568
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§ Current Affiliation: The Division of Metabolomics, China-Canada-New Zealand Joint Laboratory of
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Maternal and Fetal Medicine.
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[email protected]
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Abstract
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Pterygium is a triangular shaped ocular fibrous surface lesion growing from conjunctiva towards central
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cornea, causing ocular irritation, astigmatism, and visual disturbance. The condition is characterized by
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epithelial proliferation, fibrovascular growth, chronic inflammation, and prominent extracellular matrix
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remodeling. Studies have suggested that aberrant extracellular proteins secreted by fibroblasts lead to
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abnormal matrix production and tissue invasion contributing to the development of the disease. In this
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study, secreted proteins collected from paired pterygium and conjunctival fibroblasts in vitro were
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identified and quantified by LC-MS iTRAQ-based analysis, in which 433 proteins common to all samples
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were identified. Among these proteins, 48.0% (208) were classified as classically secreted proteins,
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17.1% (74) were exported out of the cells via non-classical secretion pathways, and 31.2% (135) were
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exosome proteins. A minority (3.7%) was not previously known to be secreted, or might be
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contaminants. 31 and 27 proteins were found up- or down-regulated in the conditioned media of
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pterygium fibroblasts relative to the media of control cells, respectively. Molecular function analysis
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showed that these proteins either belonged to catalytic proteins, structural molecules or were involved
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with receptor activities and protein binding. Further pathway analysis revealed that these proteins were
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involved in ECM-receptor interaction, focal adhesion, cancer-related, p53 signaling, complement and
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coagulation, and TGF-beta signaling pathways. These molecules identified may serve as extracellular
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ligands to activate intracellular pathways, possibly serving as potential therapeutic targets.
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Keywords: Pterygium, Fibroblast, Secretomics, iTRAQ, LC-MS/MS, Quantitative proteomics
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Introduction
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A pterygium is a superficial, external ocular mass that usually forms over the perilimbal conjunctiva and
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extends onto the corneal surface. Pterygia can vary from small, atrophic quiescent lesions to large,
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aggressive, rapidly growing fibrovascular lesions that can distort the corneal topography, and in
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advanced cases, they can obscure the optical center of the cornea (Coroneo et al., 1999; Hill and Maske,
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1989). The exact cause is unknown. However, risk factors associated with the condition include
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increased exposure to ultraviolet light, engaging in occupations that require outdoor activities, and a
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genetic predisposition to the development of pterygia (Blum, 1959). The pathophysiology of pterygia is
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characterized by elastotic degeneration of collagen (actinic elastosis) and fibrovascular proliferation,
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with an overlying covering of epithelium, chronic inflammation and aberrant extracellular matrix (ECM)
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remodeling (Bradley et al., 2010; Chui et al., 2011; Chui et al., 2008; Di Girolamo et al., 2004; Kivela and
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Uusitalo, 1998). Currently, the only effective management method for pterygium is surgical excision.
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ECM proteins do not only provide structural and biochemical support for cells to facilitate cell adhesion
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and migration, but are also involved in many other processes, such as embryogenesis, angiogenesis,
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wound healing, and cancer development (Barker, 2011; Bonnans et al., 2014). Altered expression and/or
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regulation of ECM proteins and ECM receptors have been observed in pterygium. Fibronectin, collagen I
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and III, versican, secreted protein acidic and rich in cysteine (SPARC) have been reported to be up-
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regulated in pterygium (John-Aryankalayil et al., 2006; Naib-Majani et al., 2004; Seet et al., 2012). Perez-
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Rico, et al. found that tropoelastin and elastin metabolism related genes lysyl oxidase-like 1 (LOXL-1),
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fibulin-5, fibulin-2, fibulin-3, and fibrillin-1 were over-expressed in the sub-epithelial connective tissue of
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pterygium (Perez-Rico et al., 2014; Perez-Rico et al., 2011). It has been hypothesized that dysregulation
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of elastin synthesis is involved in the pathology of the disease. Aberrant ECM molecules secreted by
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dysregulated fibroblasts may contribute to the pathology of pterygium formation by causing abnormal
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matrix protein deposition and remodeling as well as altering cell migration and proliferation. Matrix
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metalloproteinases (MMPs) are one class of several protease enzymes that modify or degrade the ECM,
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which has been previously reported in pterygium to be a class of abnormal extracellular molecules (An
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et al., 2011; Di Girolamo et al., 2001; Schellini et al., 2006; Seet et al., 2012; Solomon et al., 2000; Tsai et
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al., 2010; Yang et al., 2009). MMP-1,2,3,7,9,10 and MMP inhibitors TIMP-1,2 were expressed in
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pterygium but not in the normal conjunctiva. Imbalance of MMPs and TIMPs may result in extensive
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matrix turnover and infiltration. This has the potential to alter ECM composition and structure by
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affecting the releasing of biologically active molecules, such as growth factors.
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Most clinical studies, including previous studies of pterygium, employ immunological assays to quantify
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the protein expression. A small number of selected proteins are measured and thus do not make use of
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the information of the whole proteome. Mass spectrometry (MS) has become an indispensable tool to
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quantify the relative abundance of proteins among biological samples. Methodologies have been
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developed for such purpose, providing an effective means to analyze protein expression or post-
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translational modifications as a function of biological perturbation (Law, 2013; Law and Han, 2015).
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Although truly unbiased global profiling of the whole proteome is not currently achievable (Thompson et
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al., 2012), largely due to the complexity of the proteome, and the duty cycle of the mass spectrometers,
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these methods still offer significant advantages over conventional biochemical assays and a large
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number of proteins are concurrently monitored (Hale, 2013). Isobaric labeling-based relative
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quantification of peptides has been one of the most commonly used quantitative proteomics
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approaches with isobaric mass tags-based methods, such as iTRAQ and TMT, being the most popular
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(Rauniyar and Yates, 2014). This is because these methods are adaptable to different biological matrices,
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from biofluids, tissue extracts, cell lysates, to extracellular media, and are chemically amenable to most
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types of protein and peptide, provided they can be solubilized in a buffer stable under prolong heating.
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In our previous study, we have relatively quantified cellular proteins of pterygium and uninvolved
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conjunctival tissues from four patients using an iTRAQ approach (Hou et al., 2014). A challenge of the
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study was that the quantity of the tissue samples acquired from surgical excisions was limited and only
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25 µg of proteins from each sample was available for the analysis. This restricted our ability to
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confidently identify the proteins extracted from the tissue and quantify their expressions. In this study,
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we chose to culture primary fibroblast cells from paired pterygium and uninvolved conjunctival tissues.
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The proteins exported or secreted by the cells to the culture media were profiled and quantified. The
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secreted proteome has lower complexity than the cellular proteome and is critical to the pathology of
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the disease. Using an iTRAQ-based secretomics approach, we identified dysregulated secreted proteins
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and their possible associations with the development of pterygium.
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Methods and Materials
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Isolation of human tissues
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This research was approved by the Institutional Review Board of the Singapore Eye Research Institute
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and the procedures adhered to the tenets of the Declaration of Helsinki. Written informed consent was
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obtained from all participating patients. Patients were Chinese male at the age between 64 and 66.
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Primary nasal pterygium and uninvolved conjunctival tissues from the superior temporal quadrant of the
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same eye were collected from patients under pterygium excision surgery. Tissues were put into
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DMEM/F12 medium with 1x antibiotic/antimycotic (Life Technologies, Singapore) immediately after
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surgery, and transferred to the laboratory on ice.
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Tissue culture and production of conditioned medium
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Primary pterygium and conjunctival fibroblasts were cultured as previously described (Hou et al., 2014;
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Hou et al., 2013). All patients involved had nasal pterygia, that were all primary pterygia and moderate
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vascularity according to Donald Tan’s grading system (Tan et al., 2000). Conjunctival tissues were from
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the inferior temporal bulbar conjunctiva of the same eye. Primary fibroblast cells at passage 3-5 were
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used in this study. Conditioned medium preparation from primary pterygium and conjunctival fibroblast
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cells followed protocol previously published (See et al., 2014). Briefly, primary fibroblast cells were
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initially cultured to approximately 80% confluence with DMEM/F12 containing 10% fetal bovine serum
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(life technologies, Singapore) at 37°C in an incubator with 5% CO2. Cells were rinsed five times with
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phosphate buffered saline (PBS), and 7 milliliters of protein-free and phenol red-free DMEM/F12
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medium was added to the cells. After 24 hours of starvation, the media containing the secreted proteins
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were collected and centrifuged at 1000g for 5 minutes to remove floating cells. These media were then
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filtered using 0.22 µm filter (Millipore, Bedford, MA) to remove any remaining contaminants, and
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concentrated using a 3kDa Amicon Ultra-15 centrifugal device at 15 °C for 1 hour at 5000g. Protein
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concentration was subsequently determined using the bicinchoninic acid assay (BCA) method. The
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samples (the conditioned media) were normalized/standardized using the total protein concentration.
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The number of cells was counted when seeding onto a fresh culture plate. The actual number of cells in
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the culture at the time of harvesting was not estimated.
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iTRAQ labeling, SCX fractionation and LC-MS/MS analysis
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Samples were buffer exchanged with 100 mM TEBA (provided by the iTRAQ kit) and 100 µg of protein
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for each sample was denatured, alkylated, and digested with trypsin. Samples were labeled with the
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iTRAQ reagents according to manufacturer’s protocol as follows: the three pterygium fibroblast samples
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were labeled with reagents 114, 116 and 118 respectively, and proteins from corresponding conjunctival
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fibroblasts were labeled with reagents 113, 115 and 117 (Figure 1). All labeled samples were then
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combined into a single tube for fractionation.
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The labeled samples were cleaned and fractioned by SCX chromatography on a 1260 Infinity High-
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Performance Liquid Chromatography (Agilent Technologies, USA) equipped with a fractionator using an
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Agilent Zorbax 300-SCX column (4.6 mm × 250 mm, 5 µm). Buffer A was 5 mM potassium phosphate
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(Sigma-Aldrich) and 25% acetonitrile and buffer B was 5 mM potassium phosphate, 350 mM potassium
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chloride (Sigma-Aldrich), and 25% acetonitrile. The pH of the buffers was adjusted to 3 with phosphoric
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acid. The flow rate was set to 1.0 mL/min. 40 fractions were collected in a 60 min solvent gradient. Only
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the fractions containing the labeled peptides, determined by the UV absorptivity at 254 nm, were
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further lyophilized. The dried samples were suspended in 5% acetonitrile in 1% formic acid and were
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desalted with Pierce C-18 Spin Columns (Thermo Scientific). The cleaned fractions were lyophilized again
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and then stored at -30 °C prior to mass spectrometric analysis.
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MS analysis was performed on a QStar Elite Hybrid ESI Quadrupole time of flight tandem mass
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spectrometer (Applied Biosystems, USA) coupled to an online capillary liquid chromatography system
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(Tempo LC, Applied Biosystems, USA) with an autosampler and a peptide trapping system. Each cleaned
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peptide fraction was dissolved in 30 μL of Buffer A (2% acetonitrile in 0.1% formic acid), and 20 μL was
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injected into the system (given that the residual volume was 10 μL). The nanoLC flow was first directed
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to the peptide trap for further cleanup. The peptide trap was then switched in-line with a Thermo
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Scientific PepMap C-18 RP capillary column (150 μm x 100 mm, 3 μm, 300 Å) with a flow rate of 300
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nL/min. Peptides were eluted from the column using a 120 min linear solvent gradient. The nanoLC
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eluent was subject to positive ion nano-electrospray ionization. Data was acquired under information-
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dependent acquisition mode (IDA). The survey scan was acquired (m/z 400–1600, 0.5 seconds) with the
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five most intense multiply charged ions (counts ≥ 25) sequentially subjected to MS/MS sequencing.
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MS/MS spectra were acquired in the mass range m/z 100–2000. A dynamic exclusion was set for a
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period of 60 s. The collision energy was software determined (automatically set according to the
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precursor m/z and charge state).
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Database searches
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Database search and relative analysis were performed with the ProteinPilotTM software (AB Sciex)
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version 4.0.8085, rev 148085 with the Paragon AlgorithmTM version 4.0.0.0, rev 148083 using
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UniProtKB/Swiss-Prot database release 2013_12 (containing 49,243,530 entries) against the human
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genome (20274 sequences) (Boeckmann et al., 2003; Shilov et al., 2007). Database search was
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performed by setting cysteine modification by MMTS as a fixed modification and trypsin as the digesting
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enzyme. Bias correction was selected. The detected protein threshold (unused ProtScore) was set as
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larger than 1.3. FDR (False discovery rate) analysis was selected. Protein level changes were compared
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between paired samples of pterygium and conjunctiva. An iTRAQ fold change of ≥ 1.3 was considered to
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be up-regulated, while a ratio of ≤ 0.77 was considered down-regulated in pterygium.
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Bioinformatics and annotations
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The potential secretion pathways of proteins were predicted with the SecretomeP server 2.0 (Bendtsen
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et al., 2004a; Bendtsen et al., 2005), SignalP 4.0 (Bendtsen et al., 2004b; Petersen et al., 2011) and
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ExoCarta (Mathivanan et al., 2012; Mathivanan and Simpson, 2009) for classical and non-classical
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secretion. Protein sequences were retrieved from the UniProt database and were uploaded onto the
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SecretomeP 2.0 server for ab initio prediction of protein secretion. Gene Ontology (GO) annotation of
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proteins was performed using the online bioinformatics tool Database for Annotation, Visualization and
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Integrated Discovery (DAVID) (Huang da et al., 2009a, b). Functional annotation of proteins was
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performed using Protein ANalysis THrough Evolutionary Relationships (PANTHER) (Mi et al., 2013a; Mi et
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al., 2013b; Mi and Thomas, 2009).
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Western blot
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Western blots were performed as described previously (Hou et al., 2013). In brief, 15 µg of secreted
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proteins from primary cultured pterygium and conjunctival fibroblasts were electrophoresed under
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reducing conditions in 10-12% SDS-polyacrylamide gels. The proteins were then transferred to
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polyvinylidene difluoride (PVDF) membranes at 4°C for overnight. The membranes were blocked with
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5% milk (Sigma-Aldrich, Singapore) in TBST (Tris-Buffered Saline with 0.1% Tween-20) for 1hr at room
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temperature, subsequently incubated with primary antibodies to Fibulin-1 (Abcam, UK, AB54652,
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1:1000) or SERPINE1 (Santa Cruz, USA, SC-8979, 1:500) for 2 hrs at room temperature. Membranes were
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then incubated with secondary antibodies conjugated to horseradish peroxidase for 1 hr at room
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temperature (Sigma-Aldrich, Singapore, A9044 and A0545, 1:8000). Immunoreactivity bands were
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visualized with chemiluminescence substrate SuperSignal West Pico or West Dura (Thermal Fisher
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Scientific, Singapore).
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Immunoflurescence staining
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(Sakura® Finetek, USA) and sectioned with a Microm HM550 cryostat (Microm, Walldorf, Germany) at 8
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μm thickness. Tissue sections were fixed with 4% paraformaldehyde for 20 min, permeated in PBS
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containing 0.15% Triton X-100 for 15min, and then blocked with 4% BSA in PBS containing 0.1% Triton X-
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100 (Sigma-Aldrich) for 1hr. The blocked sections were incubated with primary antibody Fibulin-1
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(Abcam, UK, Cat. No. AB54652, 1:200 dilution in blocking solution) or SERPINE1 (Santa Cruz
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Biotechnology, USA, Cat. No. SC-8979, 1:200 dilution in blocking solution) at 4°C for overnight, washed
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three times with PBS containing 0.1% Tween-20, 5min/time. After that, tissue sections were incubated
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with Alexa Fluor 488-conjugated secondary antibody at room temperature for 45min, washed three
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times with PBS containing 0.1% Tween-20, 5min/time. Last, slides were mounted with UltraCruz
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Mounting Medium (Santa Cruz). Sections were observed and imaged using a Zeiss Axioplan 2
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fluorescence microscope (Zeiss, Oberkochen, Germany).
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Pterygium and conjunctival tissues were embedded in Optimal Cutting Temperature compound
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Results and Discussion
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iTRAQ analysis
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The iTRAQ technique has been successfully employed to study the secretomes of, for example, human
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adipose-derived cells (Blaber et al., 2012), metastatic hepatocellular carcinoma cell lines (Yu et al.,
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2013), and gastric cancer cell lines (Loei et al., 2012). The method was applied to study the secretome of
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paired pterygium and conjunctival fibroblasts surgically removed from the same patient and then 8
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cultured in vitro in this study. 433 proteins were identified in all pairs of samples by the software. The
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complete raw data from the iTRAQ analyses are shown in Supplementary Table 1. The number of
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proteins identified was consistent with the type of QStar LC-MS systems used in the previous studies.
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This justified the result for further data analysis.
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Using web-based software tools and databases (SecretomeP 2.0, SignalP 4.0, and ExoCarta), these 433
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proteins were further characterized, as classically or non-classically secreted proteins. The majority of
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the identified proteins were identified as extracellular proteins, suggesting that the samples were
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properly prepared with minimal contamination. 48.0% or 208 proteins were predicted as secreted
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proteins since they have a signal peptide within their amino acid sequence, which is a typical feature of
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classically secreted proteins. 31.2% or 135 proteins were exosome proteins, and 17.1% or 74 proteins
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were likely exported into the ECM via non-classical secretion pathways. The rest (3.7%) was non-
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secreted or were contaminants (Figure 2A). These non-secreted proteins were thought liberated in the
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culture medium upon lysis of dead cells.
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The relative quantification showed that among the 433 proteins identified, 135 proteins were
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significantly different in at least one pair of samples. These 135 proteins were then analyzed by the
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online bioinformatics tool DAVID. Part of the gene ontology analysis results are shown in Supplementary
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Table 2. Most of these proteins belong to extracellular proteins. It is consistent with the protein
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preparation method of this experiment. Pathway analysis revealed that these proteins are mostly
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involved in focal adhesion, ECM-receptor interaction, integrin signaling pathways and hemostasis (Table
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1). We considered that a protein as up- or down-regulated in pterygium if the protein was significantly
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up- or down-regulated in at least 2 out of 3 pairs of samples. We found that 31 proteins were
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significantly up-regulated while 27 proteins were down-regulated. The remaining 77 proteins were not
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clear with our experimental data (Figure 2B).
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The dysregulated proteins (58 proteins in total) are listed in Table 2. Molecular function analysis
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performed by online software tool PANTHER showed that both up- and down-regulated proteins belong
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to catalytic molecules, structural molecules, proteins involved in receptor activities and binding proteins
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(Figure 3). Further pathway analysis using the dysregulation protein list revealed that these proteins are
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involved in ECM-receptor interaction pathway, focal adhesion, pathways in cancer, p53 signaling
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pathway, complement and coagulation cascades and TGF-beta signaling pathway. There is overlapping
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of the protein lists involved in these pathways, for example, proteins involved in ECM-receptor
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interaction pathway and focal adhesion are the same. Table 3 listed different pathways and proteins
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involved.
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The associations between collagens and pterygium
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Collagen has been documented to be one of the major components of ECM. Immunohistochemistry
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studies of collagen in pterygium have produced controversial findings. Dake, et al. found that pterygium
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and normal conjunctival stroma contained collagen types I, II, III and IV (Dake et al., 1989). However,
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another group observed weak positive staining of type III and IV in pterygium stroma and were unable
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to detect types I, II and VII (Naib-Majani et al., 2004). The iTRAQ results showed that in addition to
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collagen types I, III, IV, another 6 types of collagens were detected (Supplementary Table 3), including
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collagen type V, VI, VII, VIII, XII, XVIII. The discrepancy of pterygium collagen types among these studies
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could be attributed to the detection methods and a variety of sample selections. Previous studies
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performed immunostaining using a targeted approach with commercially available antibodies, which
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might not extend to all types of collagens. This study used a MS-based discovery approach, which did
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not involve the use of antibodies. Secondly, the other studies used pterygium tissues, whereas our study
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used conditioned media from primary cultured fibroblast cells. Thirdly, in studies using tissues, collagens
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would be deposited within the tissue; in current study, collagens produced by cells could be deposited
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onto the plastic culture plates and therefore not always detected by MS. Elevated collagen type I in
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pterygium stroma has been documented in a previous study (Seet et al., 2012). The results showed that
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the collagen type Iα2, IVα2, Vα2, XIIα1 and three chains of collagen VI, α1, 2 and 3, were up-regulated in
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the conditioned media of pterygium fibroblast cells. Collagen VI contributes structural support for cells
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and enrichment of growth factors, cytokines, and other ligands on cell surfaces. Increased stromal
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expression of collagen VI has been correlated with various aspects of tumorigenesis and malignant
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progression (Park and Scherer, 2012). It is possible that over-production of collagen type VI by
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pterygium fibroblast may facilitate pterygium growth as well. However, how collagen VI and other
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collagens contribute to pterygium formation or progress needs further exploration.
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The up-regulation of Fibulin-1 and down-regulation of SERPINE1 were validated by Western
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blot analysis
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Fibulins are important for elastic matrix fibre assembly and function, and fibulin-5, fibulin-2, fibulin-3
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have been reported over-expressed in human pterygium and involved in the pathology of the disease
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(Argraves et al., 2003; Perez-Rico et al., 2011). In our previous study, transcript and protein expression
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of SERPINA1 were found down-regulated in the pterygium tissue relative to the paired conjunctival
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tissue (Hou et al., 2014). In this study, we detected that fibulin-1 is up-regulated and SERPINE1 is down-
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regulated in the culture media of pterygium fibroblasts (Table 2). These two proteins were chosen for
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further analysis by Western blot.
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Conditioned media were collected from a separate set of pterygium and conjunctival fibroblast cells. As
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in the iTRAQ experiment, proteins extracted from 6 pairs of samples acquired from the same patients
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were concentrated from the conditioned media and were analyzed by Western blot analysis (Figure 4A).
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To give a straightforward view of expression levels of proteins, the density of each band was plotted
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(Figure 4B). Fibulin-1 was found over-expressed in 4 out 6 pairs of samples, and down-regulated in 2
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pairs of samples. This reflected a between-subject variation, consistent with the results of iTRAQ relative
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quantification by mass spectrometry (Table 1). The patients’ pterygia were all T2 stage according to the
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classification system (Tan et al., 2000). The between-subject differences observed were likely caused by
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the intrinsic differences between the patients, and the subtle differences in their clinical characteristics,
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rather than being at different stage of the disease. In all 6 pairs of the samples, SERPINE1 protein level
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was lower in pterygium fibroblast than in conjunctival fibroblast cells. The expression of SERPINE1 did
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not show a large between-subject variation as in the case of Fibulin-1 expression.
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Fibulins are a family of extracellular glycoproteins, which play an important role in elastic fiber assembly.
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Over-expressed Fibulin-2, -3 and -5 were detected in the subepithelial tissues of pterygium (Perez-Rico
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et al., 2014; Perez-Rico et al., 2011). Intriguingly, in the present study, Fibulin-1 was identified as one of
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the up-regulated proteins by mass spectrometry, and further by Western blot, in which over-expression
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of Fibulin-1 was observed in 4 out of 6 pairs of samples, reflecting between-subject variations.
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Moreover, Fibulin-5 was also over-expressed in pterygium fibroblast conditioned media by mass
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spectrometry, although the change was not statistically significant (Supplementary Table 1). This may
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also due to between-subject variations, which has been shown in previous studies (Perez-Rico et al.,
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2014; Perez-Rico et al., 2011).
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SERPINE1 (also known as plasminogen activator inhibitor-1 (PAI-1)), is a serine protease inhibitor that
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principally inactivates tissue plasminogen activator (tPA) and urokinase (uPA). tPA/ uPA are the
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activators of plasminogen. Plasminogen is an inactive form of the serine protease plasmin. Activation of
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plasmin triggers a proteolysis cascade that contributes to fibrinolysis and ECM degradation. Increased
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uPA activity has been noticed in both pterygium and their fibroblasts, and the down-regulation of
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SERPINE1 observed in this study is consistent with previous reports (Chao et al., 2011). It is proposed
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that the down-regulation of SERPINE1 in pterygium cells leads to the activation of the uPA/uPAR-
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mediated signaling pathway, resulting in ECM changes, activation of integrin signaling pathway, and
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pterygium development.
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Both the mass spectrometry data and western blot data of Fibulin-1 and SERPINE1 are from fibroblast
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cells. It would be interesting to know the expression of these proteins in pterygium and conjunctiva
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tissues. Fibulin-1 was expressed in all layers of pterygium epithelium, stronger expression was observed
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in super epithelial and several layers of basal epithelial cells, indicating a basal epithelial cell
304
proliferation (Supplementary Figure 1A). In conjunctiva tissues, Fibulin-1 was also expressed in all layers
305
of epithelium, with a stronger expression in single layer of basal epithelial cells. SERPINE1 was
306
expressed in all epithelium layers of both pterygium and conjunctiva tissues, with a stronger expression
307
in basal epithelial cells (Supplementary Figure 1B). Here we have to point out that antibody specificity
308
validation for immunofluorescence staining in pterygium and conjunctival tissues was not performed.
309
Conclusions
310
In this study, we used an untargeted LC-MS approach based on the iTRAQ technique to investigate the
311
changes of the secreted proteome from the conditioned media of paired pterygium and normal
312
conjunctival fibroblast cells originated from the same patients. The pair-wise approach could potentially
313
identify changes of protein expression on individual-based, without the interferences of between-
314
subject variations, or even to determine the reasons causing the variations amount the diseased
315
subjects. Unfortunately, not all the clinical data and patient characteristics were made available to us.
316
We could only focus our efforts on identifying the common pathophysiological responses amount the
317
subjects being studied. Therefore, the intrinsic variations between the subjects became a challenge in
318
this study. This issue could be overcome with a reasonable cohort size and suitable statistical
319
approaches. However, the iTRAQ approach was not suited for population-based study because it
320
permitted only a relatively small number of samples to be analyzed in an experiment. Moreover, unlike
321
the previous studies on secreted proteins profiling on adipocytes or established cancer cell lines, the
322
paired pterygium and conjunctival primary fibroblasts were difficult to culture, as the conjunctival
323
tissues were small and outgrowth of fibroblasts was not always successful from explants. Although the
324
paired pterygium and conjunctival fibroblasts might not be in their native states, they should have
325
retained their differences, provided they were treated under an appropriate experimental condition.
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Were we able to improve our experimental design of the study by culturing the cells on collagen-coated
327
plates, or physiological mimicking environments using bioreactors (3D cell culture), a more accurate
328
description of the differences between pterygia and conjunctiva could be attained. Despite the
329
limitations, our iTRAQ analysis identified 433 proteins that were common to all pairs of samples with
330
95% confidence after the conditioned media was purified and enriched. Bioinformatics analysis showed
331
that 48% of these proteins were classically secreted proteins, 17% were non-classically secreted proteins
332
and 31% were exosome proteins. Furthermore, relative quantification found that 31 and 27 proteins
333
were either significantly up- or down-regulated. It is known that the dysregulated proteins are involved
334
in the ECM-receptor interaction, focal adhesion, cancer, p53 signaling, complement, coagulation and
335
TGF-beta signaling pathways. Of the 433 proteins identified, 58 proteins were dysregulated secreted
336
proteins, and they were mainly ECM proteins, growth factor binding proteins, proteases, and protease
337
inhibitors, as well as proteins involved in catalysis, structural and receptor binding functions. Since this
338
in vitro study was conducted using cultured cells, we could not exclude the possibility that some findings
339
might not be recapitulated in actual tissues. Furthermore, sample normalization using the total protein
340
concentration might introduce some artificial effects on quantification. An increase concentration of one
341
protein would unlikely accompany by a decrease concentration of another in physiological condition.
342
Secreted proteins are attractive drug targets due to their accessibility. It is, therefore, imperative to
343
examine the secreted proteome further for translational medicine of complex diseases, such as
344
pterygium. Emerging label-free and targeted quantitative proteomics strategies would be more
345
appropriate for the future studies.
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346 Acknowledgement
348
This research is supported by the Singapore National Research Foundation under its
349
NMRC/CSA/045/2012, NMRC/BNIG/1075/2012 and administered by the Singapore Ministry of Health’s
350
National Medical Research Council. The funders had no role in study design, data collection, and
351
analysis, decision to publish, or preparation of the manuscript. Aihua Hou would like to thank Anuradha
352
Veerappan for proofreading of the original manuscript.
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353 354
Author contributions
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ACCEPTED MANUSCRIPT
AH and MQT cultured primary pterygium and conjunctival fibroblast cells, collected conditioned medium
356
and concentrated secreted proteins. KPL designed and conducted the iTRAQ experiments, prepared the
357
samples and performed the data analysis. YPL conceived the experimental designed the study. AH, KPL
358
and LT wrote the manuscript.
359
An, M.X., Wu, K.L., Lin, S.C., 2011. Detection and comparison of matrix metalloproteinase in primary and
363
recurrent pterygium fibroblasts. Int J Ophthalmol 4, 353-356.
364
Argraves, W.S., Greene, L.M., Cooley, M.A., Gallagher, W.M., 2003. Fibulins: physiological and disease
365
perspectives. EMBO Rep 4, 1127-1131.
366
Barker, T.H., 2011. The role of ECM proteins and protein fragments in guiding cell behavior in
367
regenerative medicine. Biomaterials 32, 4211-4214.
368
Bendtsen, J.D., Jensen, L.J., Blom, N., Von Heijne, G., Brunak, S., 2004a. Feature-based prediction of non-
369
classical and leaderless protein secretion. Protein Eng Des Sel 17, 349-356.
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Bendtsen, J.D., Kiemer, L., Fausboll, A., Brunak, S., 2005. Non-classical protein secretion in bacteria. BMC
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Microbiol 5, 58.
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Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004b. Improved prediction of signal peptides:
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Blaber, S.P., Webster, R.A., Hill, C.J., Breen, E.J., Kuah, D., Vesey, G., Herbert, B.R., 2012. Analysis of in
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Blum, H.F., 1959. Carcinogenesis by Ultraviolet Light. Princeton University Press.
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Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M.C., Estreicher, A., Gasteiger, E., Martin, M.J.,
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Analyst 138, 4505-4511.
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Table 1. Pathway analysis of the 135 significantly different proteins between pterygium and conjunctiva.
RI PT
% PValue 15.67 0.000 14.93 0.000 6.72 0.065 5.97 0.022 5.22 0.001 4.48 0.001
6 5
4.48 3.73
0.002 0.067
4 3 19 5 3 19 11 11 11
2.99 2.24 14.18 3.73 2.24 14.18 8.21 8.21 8.21
0.039 0.042 0.000 0.000 0.037 0.000 0.000 0.000 0.032
10 7
7.46 5.22
0.000 0.007
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REACTOME_PATHWAY REACTOME_PATHWAY
Count 21 20 9 8 7 6
SC
KEGG_PATHWAY KEGG_PATHWAY PANTHER_PATHWAY PANTHER_PATHWAY PANTHER_PATHWAY REACTOME_PATHWAY REACTOME_PATHWAY REACTOME_PATHWAY REACTOME_PATHWAY
M AN U
KEGG_PATHWAY KEGG_PATHWAY
Term hsa04510:Focal adhesion hsa04512:ECM-receptor interaction hsa05200:Pathways in cancer hsa04810:Regulation of actin cytoskeleton hsa05222:Small cell lung cancer hsa00010:Glycolysis / Gluconeogenesis hsa04610:Complement and coagulation cascades hsa04142:Lysosome hsa05130:Pathogenic Escherichia coli infection hsa00030:Pentose phosphate pathway P00034:Integrin signaling pathway P00024:Glycolysis P00050:Plasminogen activating cascade REACT_604:Hemostasis REACT_18266:Axon guidance REACT_16888:Signaling by PDGF REACT_15380:Diabetes pathways REACT_13552:Integrin cell surface interactions REACT_474:Metabolism of carbohydrates
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Category KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY
1
ACCEPTED MANUSCRIPT
Table 2. Dysregulated proteins in conditioned media of pterygium fibroblasts.1 Accession Gene Symbol SP2
RI PT
yes
202
1.406
0.000
13.183
0.000
9.204
0.000
COL12A1
yes
81
2.291
0.000
2.312
0.000
4.130
0.000
P12109
COL6A1
yes
85
2.312
0.000
9.727
0.000
6.546
0.000
P07942
LAMB1
yes
45
1.355
0.000
4.920
0.002
5.495
0.002
Q16363
LAMA4
yes
41
P98160
HSPG2
yes
40
P09871
C1S
yes
31
P00736
C1R
yes
P12110
COL6A2
yes
Q15113
PCOLCE
yes
Q9UI42
CPA4
yes
Q4LDE5
SVEP1
P08294
SOD3
P01344
IGF2
P02545
LMNA
M AN U
SC
COL6A3
0.004
1.406
0.002
15.276
0.000
3.373
0.001
1.871
0.000
1.644
0.054
3.221
0.002
2.805
0.000
8.166
0.000
39
3.467
0.000
1.570
0.001
3.192
0.103
50
1.754
0.002
7.586
0.000
8.241
0.000
26
1.722
0.001
1.459
0.002
28.576
0.000
22
2.884
0.234
1.419
0.021
32.211
0.000
yes
18
1.472
0.541
1.706
0.001
4.246
0.002
yes
7
6.368
0.004
2.466
0.031
1.675
0.090
yes
5
3.311
0.006
12.023
0.000
3.837
0.325
Ex3
20
2.228
0.000
1.271
0.070
2.355
0.000
EP
TE D
1.380
AC C
UpP12111 regulated Q99715
Peptides 114:113 p-value 116:115 p-value 118:117 p-value (95%) 114:113 116:115 118:117
2
ACCEPTED MANUSCRIPT
FBLN1
yes
41
8.872
0.001
0.904
0.318
10.186
0.001
P24043
LAMA2
yes
14
2.754
0.002
1.096
0.708
7.244
0.003
P51884
LUM
non-classical 16
1.675
0.001
25.119
0.007
1.236
0.019
P37802
TAGLN2
non-classical 18
1.660
0.040
0.847
0.361
2.377
0.004
Q14204
DYNC1H1
ex
3
1.570
0.001
1.675
0.013
1.127
0.004
P08123
COL1A2
yes
531
1.706
0.000
0.258
0.000
1.600
0.000
P08670
VIM
non-classical 99
1.585
0.032
0.445
0.000
2.992
0.000
P18206
VCL
ex
35
1.380
0.045
0.501
0.012
2.051
0.000
Q13813
SPTAN1
ex
15
1.660
0.033
1.738
0.009
0.698
0.001
P36955
SERPINF1
yes
21
5.445
0.000
0.597
0.133
5.058
0.001
P05997
COL5A2
yes
P08572
COL4A2
yes
P35442
THBS2
yes
Q01995
TAGLN
P04264
KRT1
P51888
PRELP
DownP02751 regulated P02452
TE D
M AN U
SC
RI PT
P23142
3.631
0.004
0.122
0.004
1.959
0.005
20
1.977
0.002
0.196
0.025
2.070
0.002
14
2.270
0.007
4.246
0.001
0.441
0.006
non-classical 24
2.443
0.004
0.138
0.000
2.421
0.000
AC C
EP
26
ex
8
3.733
0.045
8.395
0.001
0.015
0.009
yes
7
3.162
0.016
17.378
0.001
0.497
0.552
FN1
yes
349
0.128
0.000
0.146
0.000
0.213
0.000
COL1A1
yes
426
0.207
0.088
0.466
0.020
0.520
0.002 3
ACCEPTED MANUSCRIPT
THBS1
yes
47
0.270
0.000
0.692
0.040
0.118
0.000
O00391
QSOX1
yes
37
0.229
0.000
0.249
0.000
0.466
0.000
P05121
SERPINE1
yes
57
0.221
0.000
0.041
0.000
0.275
0.000
Q12805
EFEMP1
yes
54
0.095
0.000
0.597
0.077
0.094
0.001
P09486
SPARC
yes
84
0.113
0.014
0.196
0.001
0.625
0.776
Q15582
TGFBI
yes
43
0.391
0.000
0.145
0.000
0.597
0.082
P01033
TIMP1
yes
40
0.229
0.119
0.316
0.022
0.036
0.000
P00338
LDHA
ex
23
0.163
0.000
0.515
0.012
0.240
0.000
Q14767
LTBP2
yes
19
0.421
0.000
0.409
0.000
0.305
0.000
Q05682
CALD1
Un4
16
0.631
0.430
0.402
0.000
0.470
0.010
P17936
IGFBP3
yes
Q08629
SPOCK1
yes
P07858
CTSB
yes
Q7Z7G0
ABI3BP
yes
P13611
VCAN
Q14766
LTBP1
P55268 Q76M96
TE D
M AN U
SC
RI PT
P07996
0.291
0.004
0.631
0.260
0.021
0.002
13
0.445
0.033
0.483
0.026
0.091
0.007
6
0.479
0.043
0.649
0.275
0.296
0.007
6
0.409
0.031
0.366
0.076
0.029
0.002
yes
8
0.213
0.011
0.064
0.083
0.018
0.030
yes
4
0.120
0.039
0.108
0.056
0.187
0.058
LAMB2
yes
40
0.912
0.502
0.679
0.000
0.029
0.000
CCDC80
yes
27
1.202
0.419
0.319
0.000
0.045
0.000
AC C
EP
16
4
ACCEPTED MANUSCRIPT
SERPINE2
yes
38
0.429
0.001
0.991
0.842
0.090
0.000
Q9UBP4
DKK3
yes
17
1.148
0.436
0.087
0.011
0.247
0.002
P00558
PGK1
ex
10
0.328
0.020
0.254
0.014
1.057
0.432
P27816
MAP4
un
4
0.752
0.015
0.802
0.137
0.319
0.004
P35555
FBN1
yes
47
0.283
0.000
0.773
0.199
0.215
0.000
P03956
MMP1
yes
26
0.511
0.000
1.854
0.008
0.016
0.000
P24592
IGFBP6
yes
11
0.299
0.038
0.366
0.030
1.343
0.602
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P07093
at least in 2 out of 3 pairs of samples, Fold changes by iTRAQ Analysis
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signaling peptides;
3
exosome;
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unknown.
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Table 3. Different pathways of which the dysregulated secreted proteins were involved in. Pathways1
Gene lists
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collagen, type I, alpha 1 collagen, type I, alpha 2 collagen, type IV, alpha 2
collagen, type VI, alpha 1
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collagen, type VI, alpha 2
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collagen, type V, alpha 2
collagen, type VI, alpha 3 fibronectin 1
ECM-receptor interaction
heparan sulfate proteoglycan 2 laminin, alpha 2
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laminin, alpha 4 laminin, beta 1
laminin, beta 2 (laminin S)
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thrombospondin 1 thrombospondin 2
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Focal adhesion
The same list as “ECM-receptor interaction” collagen, type IV, alpha 2
fibronectin 1 laminin, alpha 2
Pathways in cancer
laminin, alpha 4 laminin, beta 1 laminin, beta 2 (laminin S)
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matrix metallopeptidase 1 insulin-like growth factor binding protein 3 serpin peptidase inhibitor, clade E, member 1
p53 signaling pathway
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thrombospondin 1 complement component 1, r subcomponent Complement and coagulation cascades
complement component 1, s subcomponent
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serpin peptidase inhibitor, clade E, member 1
latent transforming growth factor beta binding protein 1 thrombospondin 1
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TGF-beta signaling pathway
thrombospondin 2
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DAVID, pathways, KEGG_PATHWAY were chosen.
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Figures and Legends
Figure 1. Pterygium secretome experimental design.
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Figure 2. (A) Pie chart showing the types of extracellular proteins identified by mass spectrometry. (B) Pie chart showing up- or down-regulated proteins in the conditioned media of pterygium fibroblasts.
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Figure 3. Bar charts showing biological function analysis results of the up- or down-regulated proteins by PANTHER.
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Figure 4. A. Protein expression of Fibulin-1 and SERPINE1 in conditioned media of pterygium and conjunctival fibroblast cells was determined by immunoblotting with antibodies specific for Fibulin-1 and SERPINE1. B. Densitometry measurement of the bands in A.
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Highlights
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Secreted proteins collected from paired pterygium and conjunctival fibroblasts were identified and quantified by iTRAQ-based tandem mass spectrometry. 433 proteins were identified, of which 31 were up-regulated and 27 were down-regulated in conditioned media of pterygium fibroblast compared to control. Molecular function analysis suggested that these proteins belonged to catalytic proteins, structural molecules or were involved with receptor activities and protein binding.
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Pathway analysis suggested that these proteins were involved in ECM-receptor interaction, focal adhesion, cancer, p53 signaling, complement, coagulation, and TGF-beta signaling pathways.
S0014-4835(16)30323-2
DOI:
10.1016/j.exer.2016.10.006
Reference:
YEXER 7034
To appear in:
Experimental Eye Research
Received Date: 9 May 2016 Revised Date:
26 September 2016
Accepted Date: 6 October 2016
Please cite this article as: Hou, A., Law, K.P., Tin, M.Q., Lim, Y.P., Tong, L., In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy, Experimental Eye Research (2016), doi: 10.1016/j.exer.2016.10.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In vitro Secretomics study of Pterygium-derived Fibroblasts by iTRAQ-
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based Quantitative Proteomics Strategy
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3 Aihua Hou 1,a, Kai Pong Law 2,a, §, Min Qi Tin 1, Yoon Pin Lim 2,5 and Louis Tong 1,3,4,5*
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Ocular Surface Research Group, Singapore Eye Research Institute, Singapore
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2
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore
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3
Duke-NUS Graduate Medical School, Singapore
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Singapore National Eye Center, Singapore
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Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore
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a
Equally contributed authors
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* Correspondence authors:
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Address: 20 College Road, The Academia, Level 6, Discovery Tower Singapore 169856, Tel: +65-
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65767213; Fax: +65- 62252568
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§ Current Affiliation: The Division of Metabolomics, China-Canada-New Zealand Joint Laboratory of
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Maternal and Fetal Medicine.
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[email protected]
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Abstract
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Pterygium is a triangular shaped ocular fibrous surface lesion growing from conjunctiva towards central
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cornea, causing ocular irritation, astigmatism, and visual disturbance. The condition is characterized by
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epithelial proliferation, fibrovascular growth, chronic inflammation, and prominent extracellular matrix
21
remodeling. Studies have suggested that aberrant extracellular proteins secreted by fibroblasts lead to
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abnormal matrix production and tissue invasion contributing to the development of the disease. In this
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study, secreted proteins collected from paired pterygium and conjunctival fibroblasts in vitro were
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identified and quantified by LC-MS iTRAQ-based analysis, in which 433 proteins common to all samples
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were identified. Among these proteins, 48.0% (208) were classified as classically secreted proteins,
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17.1% (74) were exported out of the cells via non-classical secretion pathways, and 31.2% (135) were
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exosome proteins. A minority (3.7%) was not previously known to be secreted, or might be
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contaminants. 31 and 27 proteins were found up- or down-regulated in the conditioned media of
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pterygium fibroblasts relative to the media of control cells, respectively. Molecular function analysis
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showed that these proteins either belonged to catalytic proteins, structural molecules or were involved
31
with receptor activities and protein binding. Further pathway analysis revealed that these proteins were
32
involved in ECM-receptor interaction, focal adhesion, cancer-related, p53 signaling, complement and
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coagulation, and TGF-beta signaling pathways. These molecules identified may serve as extracellular
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ligands to activate intracellular pathways, possibly serving as potential therapeutic targets.
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Keywords: Pterygium, Fibroblast, Secretomics, iTRAQ, LC-MS/MS, Quantitative proteomics
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Introduction
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A pterygium is a superficial, external ocular mass that usually forms over the perilimbal conjunctiva and
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extends onto the corneal surface. Pterygia can vary from small, atrophic quiescent lesions to large,
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aggressive, rapidly growing fibrovascular lesions that can distort the corneal topography, and in
42
advanced cases, they can obscure the optical center of the cornea (Coroneo et al., 1999; Hill and Maske,
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1989). The exact cause is unknown. However, risk factors associated with the condition include
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increased exposure to ultraviolet light, engaging in occupations that require outdoor activities, and a
45
genetic predisposition to the development of pterygia (Blum, 1959). The pathophysiology of pterygia is
46
characterized by elastotic degeneration of collagen (actinic elastosis) and fibrovascular proliferation,
47
with an overlying covering of epithelium, chronic inflammation and aberrant extracellular matrix (ECM)
48
remodeling (Bradley et al., 2010; Chui et al., 2011; Chui et al., 2008; Di Girolamo et al., 2004; Kivela and
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Uusitalo, 1998). Currently, the only effective management method for pterygium is surgical excision.
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ECM proteins do not only provide structural and biochemical support for cells to facilitate cell adhesion
51
and migration, but are also involved in many other processes, such as embryogenesis, angiogenesis,
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wound healing, and cancer development (Barker, 2011; Bonnans et al., 2014). Altered expression and/or
53
regulation of ECM proteins and ECM receptors have been observed in pterygium. Fibronectin, collagen I
54
and III, versican, secreted protein acidic and rich in cysteine (SPARC) have been reported to be up-
55
regulated in pterygium (John-Aryankalayil et al., 2006; Naib-Majani et al., 2004; Seet et al., 2012). Perez-
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Rico, et al. found that tropoelastin and elastin metabolism related genes lysyl oxidase-like 1 (LOXL-1),
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fibulin-5, fibulin-2, fibulin-3, and fibrillin-1 were over-expressed in the sub-epithelial connective tissue of
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pterygium (Perez-Rico et al., 2014; Perez-Rico et al., 2011). It has been hypothesized that dysregulation
59
of elastin synthesis is involved in the pathology of the disease. Aberrant ECM molecules secreted by
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dysregulated fibroblasts may contribute to the pathology of pterygium formation by causing abnormal
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matrix protein deposition and remodeling as well as altering cell migration and proliferation. Matrix
62
metalloproteinases (MMPs) are one class of several protease enzymes that modify or degrade the ECM,
63
which has been previously reported in pterygium to be a class of abnormal extracellular molecules (An
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et al., 2011; Di Girolamo et al., 2001; Schellini et al., 2006; Seet et al., 2012; Solomon et al., 2000; Tsai et
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al., 2010; Yang et al., 2009). MMP-1,2,3,7,9,10 and MMP inhibitors TIMP-1,2 were expressed in
66
pterygium but not in the normal conjunctiva. Imbalance of MMPs and TIMPs may result in extensive
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matrix turnover and infiltration. This has the potential to alter ECM composition and structure by
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affecting the releasing of biologically active molecules, such as growth factors.
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Most clinical studies, including previous studies of pterygium, employ immunological assays to quantify
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the protein expression. A small number of selected proteins are measured and thus do not make use of
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the information of the whole proteome. Mass spectrometry (MS) has become an indispensable tool to
72
quantify the relative abundance of proteins among biological samples. Methodologies have been
73
developed for such purpose, providing an effective means to analyze protein expression or post-
74
translational modifications as a function of biological perturbation (Law, 2013; Law and Han, 2015).
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Although truly unbiased global profiling of the whole proteome is not currently achievable (Thompson et
76
al., 2012), largely due to the complexity of the proteome, and the duty cycle of the mass spectrometers,
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these methods still offer significant advantages over conventional biochemical assays and a large
78
number of proteins are concurrently monitored (Hale, 2013). Isobaric labeling-based relative
79
quantification of peptides has been one of the most commonly used quantitative proteomics
80
approaches with isobaric mass tags-based methods, such as iTRAQ and TMT, being the most popular
81
(Rauniyar and Yates, 2014). This is because these methods are adaptable to different biological matrices,
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from biofluids, tissue extracts, cell lysates, to extracellular media, and are chemically amenable to most
83
types of protein and peptide, provided they can be solubilized in a buffer stable under prolong heating.
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In our previous study, we have relatively quantified cellular proteins of pterygium and uninvolved
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conjunctival tissues from four patients using an iTRAQ approach (Hou et al., 2014). A challenge of the
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study was that the quantity of the tissue samples acquired from surgical excisions was limited and only
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25 µg of proteins from each sample was available for the analysis. This restricted our ability to
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confidently identify the proteins extracted from the tissue and quantify their expressions. In this study,
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we chose to culture primary fibroblast cells from paired pterygium and uninvolved conjunctival tissues.
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The proteins exported or secreted by the cells to the culture media were profiled and quantified. The
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secreted proteome has lower complexity than the cellular proteome and is critical to the pathology of
92
the disease. Using an iTRAQ-based secretomics approach, we identified dysregulated secreted proteins
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and their possible associations with the development of pterygium.
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Methods and Materials
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Isolation of human tissues
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This research was approved by the Institutional Review Board of the Singapore Eye Research Institute
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and the procedures adhered to the tenets of the Declaration of Helsinki. Written informed consent was
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obtained from all participating patients. Patients were Chinese male at the age between 64 and 66.
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Primary nasal pterygium and uninvolved conjunctival tissues from the superior temporal quadrant of the
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same eye were collected from patients under pterygium excision surgery. Tissues were put into
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DMEM/F12 medium with 1x antibiotic/antimycotic (Life Technologies, Singapore) immediately after
103
surgery, and transferred to the laboratory on ice.
104
Tissue culture and production of conditioned medium
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Primary pterygium and conjunctival fibroblasts were cultured as previously described (Hou et al., 2014;
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Hou et al., 2013). All patients involved had nasal pterygia, that were all primary pterygia and moderate
107
vascularity according to Donald Tan’s grading system (Tan et al., 2000). Conjunctival tissues were from
108
the inferior temporal bulbar conjunctiva of the same eye. Primary fibroblast cells at passage 3-5 were
109
used in this study. Conditioned medium preparation from primary pterygium and conjunctival fibroblast
110
cells followed protocol previously published (See et al., 2014). Briefly, primary fibroblast cells were
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initially cultured to approximately 80% confluence with DMEM/F12 containing 10% fetal bovine serum
112
(life technologies, Singapore) at 37°C in an incubator with 5% CO2. Cells were rinsed five times with
113
phosphate buffered saline (PBS), and 7 milliliters of protein-free and phenol red-free DMEM/F12
114
medium was added to the cells. After 24 hours of starvation, the media containing the secreted proteins
115
were collected and centrifuged at 1000g for 5 minutes to remove floating cells. These media were then
116
filtered using 0.22 µm filter (Millipore, Bedford, MA) to remove any remaining contaminants, and
117
concentrated using a 3kDa Amicon Ultra-15 centrifugal device at 15 °C for 1 hour at 5000g. Protein
118
concentration was subsequently determined using the bicinchoninic acid assay (BCA) method. The
119
samples (the conditioned media) were normalized/standardized using the total protein concentration.
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The number of cells was counted when seeding onto a fresh culture plate. The actual number of cells in
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the culture at the time of harvesting was not estimated.
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iTRAQ labeling, SCX fractionation and LC-MS/MS analysis
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Samples were buffer exchanged with 100 mM TEBA (provided by the iTRAQ kit) and 100 µg of protein
124
for each sample was denatured, alkylated, and digested with trypsin. Samples were labeled with the
125
iTRAQ reagents according to manufacturer’s protocol as follows: the three pterygium fibroblast samples
126
were labeled with reagents 114, 116 and 118 respectively, and proteins from corresponding conjunctival
127
fibroblasts were labeled with reagents 113, 115 and 117 (Figure 1). All labeled samples were then
128
combined into a single tube for fractionation.
129
The labeled samples were cleaned and fractioned by SCX chromatography on a 1260 Infinity High-
130
Performance Liquid Chromatography (Agilent Technologies, USA) equipped with a fractionator using an
131
Agilent Zorbax 300-SCX column (4.6 mm × 250 mm, 5 µm). Buffer A was 5 mM potassium phosphate
132
(Sigma-Aldrich) and 25% acetonitrile and buffer B was 5 mM potassium phosphate, 350 mM potassium
133
chloride (Sigma-Aldrich), and 25% acetonitrile. The pH of the buffers was adjusted to 3 with phosphoric
134
acid. The flow rate was set to 1.0 mL/min. 40 fractions were collected in a 60 min solvent gradient. Only
135
the fractions containing the labeled peptides, determined by the UV absorptivity at 254 nm, were
136
further lyophilized. The dried samples were suspended in 5% acetonitrile in 1% formic acid and were
137
desalted with Pierce C-18 Spin Columns (Thermo Scientific). The cleaned fractions were lyophilized again
138
and then stored at -30 °C prior to mass spectrometric analysis.
139
MS analysis was performed on a QStar Elite Hybrid ESI Quadrupole time of flight tandem mass
140
spectrometer (Applied Biosystems, USA) coupled to an online capillary liquid chromatography system
141
(Tempo LC, Applied Biosystems, USA) with an autosampler and a peptide trapping system. Each cleaned
142
peptide fraction was dissolved in 30 μL of Buffer A (2% acetonitrile in 0.1% formic acid), and 20 μL was
143
injected into the system (given that the residual volume was 10 μL). The nanoLC flow was first directed
144
to the peptide trap for further cleanup. The peptide trap was then switched in-line with a Thermo
145
Scientific PepMap C-18 RP capillary column (150 μm x 100 mm, 3 μm, 300 Å) with a flow rate of 300
146
nL/min. Peptides were eluted from the column using a 120 min linear solvent gradient. The nanoLC
147
eluent was subject to positive ion nano-electrospray ionization. Data was acquired under information-
148
dependent acquisition mode (IDA). The survey scan was acquired (m/z 400–1600, 0.5 seconds) with the
149
five most intense multiply charged ions (counts ≥ 25) sequentially subjected to MS/MS sequencing.
150
MS/MS spectra were acquired in the mass range m/z 100–2000. A dynamic exclusion was set for a
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period of 60 s. The collision energy was software determined (automatically set according to the
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precursor m/z and charge state).
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Database searches
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Database search and relative analysis were performed with the ProteinPilotTM software (AB Sciex)
155
version 4.0.8085, rev 148085 with the Paragon AlgorithmTM version 4.0.0.0, rev 148083 using
156
UniProtKB/Swiss-Prot database release 2013_12 (containing 49,243,530 entries) against the human
157
genome (20274 sequences) (Boeckmann et al., 2003; Shilov et al., 2007). Database search was
158
performed by setting cysteine modification by MMTS as a fixed modification and trypsin as the digesting
159
enzyme. Bias correction was selected. The detected protein threshold (unused ProtScore) was set as
160
larger than 1.3. FDR (False discovery rate) analysis was selected. Protein level changes were compared
161
between paired samples of pterygium and conjunctiva. An iTRAQ fold change of ≥ 1.3 was considered to
162
be up-regulated, while a ratio of ≤ 0.77 was considered down-regulated in pterygium.
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Bioinformatics and annotations
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The potential secretion pathways of proteins were predicted with the SecretomeP server 2.0 (Bendtsen
165
et al., 2004a; Bendtsen et al., 2005), SignalP 4.0 (Bendtsen et al., 2004b; Petersen et al., 2011) and
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ExoCarta (Mathivanan et al., 2012; Mathivanan and Simpson, 2009) for classical and non-classical
167
secretion. Protein sequences were retrieved from the UniProt database and were uploaded onto the
168
SecretomeP 2.0 server for ab initio prediction of protein secretion. Gene Ontology (GO) annotation of
169
proteins was performed using the online bioinformatics tool Database for Annotation, Visualization and
170
Integrated Discovery (DAVID) (Huang da et al., 2009a, b). Functional annotation of proteins was
171
performed using Protein ANalysis THrough Evolutionary Relationships (PANTHER) (Mi et al., 2013a; Mi et
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al., 2013b; Mi and Thomas, 2009).
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Western blot
174
Western blots were performed as described previously (Hou et al., 2013). In brief, 15 µg of secreted
175
proteins from primary cultured pterygium and conjunctival fibroblasts were electrophoresed under
176
reducing conditions in 10-12% SDS-polyacrylamide gels. The proteins were then transferred to
177
polyvinylidene difluoride (PVDF) membranes at 4°C for overnight. The membranes were blocked with
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5% milk (Sigma-Aldrich, Singapore) in TBST (Tris-Buffered Saline with 0.1% Tween-20) for 1hr at room
179
temperature, subsequently incubated with primary antibodies to Fibulin-1 (Abcam, UK, AB54652,
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1:1000) or SERPINE1 (Santa Cruz, USA, SC-8979, 1:500) for 2 hrs at room temperature. Membranes were
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then incubated with secondary antibodies conjugated to horseradish peroxidase for 1 hr at room
182
temperature (Sigma-Aldrich, Singapore, A9044 and A0545, 1:8000). Immunoreactivity bands were
183
visualized with chemiluminescence substrate SuperSignal West Pico or West Dura (Thermal Fisher
184
Scientific, Singapore).
185 186 187
Immunoflurescence staining
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(Sakura® Finetek, USA) and sectioned with a Microm HM550 cryostat (Microm, Walldorf, Germany) at 8
189
μm thickness. Tissue sections were fixed with 4% paraformaldehyde for 20 min, permeated in PBS
190
containing 0.15% Triton X-100 for 15min, and then blocked with 4% BSA in PBS containing 0.1% Triton X-
191
100 (Sigma-Aldrich) for 1hr. The blocked sections were incubated with primary antibody Fibulin-1
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(Abcam, UK, Cat. No. AB54652, 1:200 dilution in blocking solution) or SERPINE1 (Santa Cruz
193
Biotechnology, USA, Cat. No. SC-8979, 1:200 dilution in blocking solution) at 4°C for overnight, washed
194
three times with PBS containing 0.1% Tween-20, 5min/time. After that, tissue sections were incubated
195
with Alexa Fluor 488-conjugated secondary antibody at room temperature for 45min, washed three
196
times with PBS containing 0.1% Tween-20, 5min/time. Last, slides were mounted with UltraCruz
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Mounting Medium (Santa Cruz). Sections were observed and imaged using a Zeiss Axioplan 2
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fluorescence microscope (Zeiss, Oberkochen, Germany).
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Pterygium and conjunctival tissues were embedded in Optimal Cutting Temperature compound
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Results and Discussion
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iTRAQ analysis
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The iTRAQ technique has been successfully employed to study the secretomes of, for example, human
203
adipose-derived cells (Blaber et al., 2012), metastatic hepatocellular carcinoma cell lines (Yu et al.,
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2013), and gastric cancer cell lines (Loei et al., 2012). The method was applied to study the secretome of
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paired pterygium and conjunctival fibroblasts surgically removed from the same patient and then 8
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cultured in vitro in this study. 433 proteins were identified in all pairs of samples by the software. The
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complete raw data from the iTRAQ analyses are shown in Supplementary Table 1. The number of
208
proteins identified was consistent with the type of QStar LC-MS systems used in the previous studies.
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This justified the result for further data analysis.
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Using web-based software tools and databases (SecretomeP 2.0, SignalP 4.0, and ExoCarta), these 433
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proteins were further characterized, as classically or non-classically secreted proteins. The majority of
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the identified proteins were identified as extracellular proteins, suggesting that the samples were
213
properly prepared with minimal contamination. 48.0% or 208 proteins were predicted as secreted
214
proteins since they have a signal peptide within their amino acid sequence, which is a typical feature of
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classically secreted proteins. 31.2% or 135 proteins were exosome proteins, and 17.1% or 74 proteins
216
were likely exported into the ECM via non-classical secretion pathways. The rest (3.7%) was non-
217
secreted or were contaminants (Figure 2A). These non-secreted proteins were thought liberated in the
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culture medium upon lysis of dead cells.
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The relative quantification showed that among the 433 proteins identified, 135 proteins were
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significantly different in at least one pair of samples. These 135 proteins were then analyzed by the
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online bioinformatics tool DAVID. Part of the gene ontology analysis results are shown in Supplementary
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Table 2. Most of these proteins belong to extracellular proteins. It is consistent with the protein
223
preparation method of this experiment. Pathway analysis revealed that these proteins are mostly
224
involved in focal adhesion, ECM-receptor interaction, integrin signaling pathways and hemostasis (Table
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1). We considered that a protein as up- or down-regulated in pterygium if the protein was significantly
226
up- or down-regulated in at least 2 out of 3 pairs of samples. We found that 31 proteins were
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significantly up-regulated while 27 proteins were down-regulated. The remaining 77 proteins were not
228
clear with our experimental data (Figure 2B).
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The dysregulated proteins (58 proteins in total) are listed in Table 2. Molecular function analysis
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performed by online software tool PANTHER showed that both up- and down-regulated proteins belong
231
to catalytic molecules, structural molecules, proteins involved in receptor activities and binding proteins
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(Figure 3). Further pathway analysis using the dysregulation protein list revealed that these proteins are
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involved in ECM-receptor interaction pathway, focal adhesion, pathways in cancer, p53 signaling
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pathway, complement and coagulation cascades and TGF-beta signaling pathway. There is overlapping
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of the protein lists involved in these pathways, for example, proteins involved in ECM-receptor
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interaction pathway and focal adhesion are the same. Table 3 listed different pathways and proteins
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involved.
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The associations between collagens and pterygium
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Collagen has been documented to be one of the major components of ECM. Immunohistochemistry
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studies of collagen in pterygium have produced controversial findings. Dake, et al. found that pterygium
241
and normal conjunctival stroma contained collagen types I, II, III and IV (Dake et al., 1989). However,
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another group observed weak positive staining of type III and IV in pterygium stroma and were unable
243
to detect types I, II and VII (Naib-Majani et al., 2004). The iTRAQ results showed that in addition to
244
collagen types I, III, IV, another 6 types of collagens were detected (Supplementary Table 3), including
245
collagen type V, VI, VII, VIII, XII, XVIII. The discrepancy of pterygium collagen types among these studies
246
could be attributed to the detection methods and a variety of sample selections. Previous studies
247
performed immunostaining using a targeted approach with commercially available antibodies, which
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might not extend to all types of collagens. This study used a MS-based discovery approach, which did
249
not involve the use of antibodies. Secondly, the other studies used pterygium tissues, whereas our study
250
used conditioned media from primary cultured fibroblast cells. Thirdly, in studies using tissues, collagens
251
would be deposited within the tissue; in current study, collagens produced by cells could be deposited
252
onto the plastic culture plates and therefore not always detected by MS. Elevated collagen type I in
253
pterygium stroma has been documented in a previous study (Seet et al., 2012). The results showed that
254
the collagen type Iα2, IVα2, Vα2, XIIα1 and three chains of collagen VI, α1, 2 and 3, were up-regulated in
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the conditioned media of pterygium fibroblast cells. Collagen VI contributes structural support for cells
256
and enrichment of growth factors, cytokines, and other ligands on cell surfaces. Increased stromal
257
expression of collagen VI has been correlated with various aspects of tumorigenesis and malignant
258
progression (Park and Scherer, 2012). It is possible that over-production of collagen type VI by
259
pterygium fibroblast may facilitate pterygium growth as well. However, how collagen VI and other
260
collagens contribute to pterygium formation or progress needs further exploration.
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The up-regulation of Fibulin-1 and down-regulation of SERPINE1 were validated by Western
262
blot analysis
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Fibulins are important for elastic matrix fibre assembly and function, and fibulin-5, fibulin-2, fibulin-3
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have been reported over-expressed in human pterygium and involved in the pathology of the disease
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(Argraves et al., 2003; Perez-Rico et al., 2011). In our previous study, transcript and protein expression
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of SERPINA1 were found down-regulated in the pterygium tissue relative to the paired conjunctival
267
tissue (Hou et al., 2014). In this study, we detected that fibulin-1 is up-regulated and SERPINE1 is down-
268
regulated in the culture media of pterygium fibroblasts (Table 2). These two proteins were chosen for
269
further analysis by Western blot.
270
Conditioned media were collected from a separate set of pterygium and conjunctival fibroblast cells. As
271
in the iTRAQ experiment, proteins extracted from 6 pairs of samples acquired from the same patients
272
were concentrated from the conditioned media and were analyzed by Western blot analysis (Figure 4A).
273
To give a straightforward view of expression levels of proteins, the density of each band was plotted
274
(Figure 4B). Fibulin-1 was found over-expressed in 4 out 6 pairs of samples, and down-regulated in 2
275
pairs of samples. This reflected a between-subject variation, consistent with the results of iTRAQ relative
276
quantification by mass spectrometry (Table 1). The patients’ pterygia were all T2 stage according to the
277
classification system (Tan et al., 2000). The between-subject differences observed were likely caused by
278
the intrinsic differences between the patients, and the subtle differences in their clinical characteristics,
279
rather than being at different stage of the disease. In all 6 pairs of the samples, SERPINE1 protein level
280
was lower in pterygium fibroblast than in conjunctival fibroblast cells. The expression of SERPINE1 did
281
not show a large between-subject variation as in the case of Fibulin-1 expression.
282
Fibulins are a family of extracellular glycoproteins, which play an important role in elastic fiber assembly.
283
Over-expressed Fibulin-2, -3 and -5 were detected in the subepithelial tissues of pterygium (Perez-Rico
284
et al., 2014; Perez-Rico et al., 2011). Intriguingly, in the present study, Fibulin-1 was identified as one of
285
the up-regulated proteins by mass spectrometry, and further by Western blot, in which over-expression
286
of Fibulin-1 was observed in 4 out of 6 pairs of samples, reflecting between-subject variations.
287
Moreover, Fibulin-5 was also over-expressed in pterygium fibroblast conditioned media by mass
288
spectrometry, although the change was not statistically significant (Supplementary Table 1). This may
289
also due to between-subject variations, which has been shown in previous studies (Perez-Rico et al.,
290
2014; Perez-Rico et al., 2011).
291
SERPINE1 (also known as plasminogen activator inhibitor-1 (PAI-1)), is a serine protease inhibitor that
292
principally inactivates tissue plasminogen activator (tPA) and urokinase (uPA). tPA/ uPA are the
293
activators of plasminogen. Plasminogen is an inactive form of the serine protease plasmin. Activation of
294
plasmin triggers a proteolysis cascade that contributes to fibrinolysis and ECM degradation. Increased
295
uPA activity has been noticed in both pterygium and their fibroblasts, and the down-regulation of
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SERPINE1 observed in this study is consistent with previous reports (Chao et al., 2011). It is proposed
297
that the down-regulation of SERPINE1 in pterygium cells leads to the activation of the uPA/uPAR-
298
mediated signaling pathway, resulting in ECM changes, activation of integrin signaling pathway, and
299
pterygium development.
300
Both the mass spectrometry data and western blot data of Fibulin-1 and SERPINE1 are from fibroblast
301
cells. It would be interesting to know the expression of these proteins in pterygium and conjunctiva
302
tissues. Fibulin-1 was expressed in all layers of pterygium epithelium, stronger expression was observed
303
in super epithelial and several layers of basal epithelial cells, indicating a basal epithelial cell
304
proliferation (Supplementary Figure 1A). In conjunctiva tissues, Fibulin-1 was also expressed in all layers
305
of epithelium, with a stronger expression in single layer of basal epithelial cells. SERPINE1 was
306
expressed in all epithelium layers of both pterygium and conjunctiva tissues, with a stronger expression
307
in basal epithelial cells (Supplementary Figure 1B). Here we have to point out that antibody specificity
308
validation for immunofluorescence staining in pterygium and conjunctival tissues was not performed.
309
Conclusions
310
In this study, we used an untargeted LC-MS approach based on the iTRAQ technique to investigate the
311
changes of the secreted proteome from the conditioned media of paired pterygium and normal
312
conjunctival fibroblast cells originated from the same patients. The pair-wise approach could potentially
313
identify changes of protein expression on individual-based, without the interferences of between-
314
subject variations, or even to determine the reasons causing the variations amount the diseased
315
subjects. Unfortunately, not all the clinical data and patient characteristics were made available to us.
316
We could only focus our efforts on identifying the common pathophysiological responses amount the
317
subjects being studied. Therefore, the intrinsic variations between the subjects became a challenge in
318
this study. This issue could be overcome with a reasonable cohort size and suitable statistical
319
approaches. However, the iTRAQ approach was not suited for population-based study because it
320
permitted only a relatively small number of samples to be analyzed in an experiment. Moreover, unlike
321
the previous studies on secreted proteins profiling on adipocytes or established cancer cell lines, the
322
paired pterygium and conjunctival primary fibroblasts were difficult to culture, as the conjunctival
323
tissues were small and outgrowth of fibroblasts was not always successful from explants. Although the
324
paired pterygium and conjunctival fibroblasts might not be in their native states, they should have
325
retained their differences, provided they were treated under an appropriate experimental condition.
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Were we able to improve our experimental design of the study by culturing the cells on collagen-coated
327
plates, or physiological mimicking environments using bioreactors (3D cell culture), a more accurate
328
description of the differences between pterygia and conjunctiva could be attained. Despite the
329
limitations, our iTRAQ analysis identified 433 proteins that were common to all pairs of samples with
330
95% confidence after the conditioned media was purified and enriched. Bioinformatics analysis showed
331
that 48% of these proteins were classically secreted proteins, 17% were non-classically secreted proteins
332
and 31% were exosome proteins. Furthermore, relative quantification found that 31 and 27 proteins
333
were either significantly up- or down-regulated. It is known that the dysregulated proteins are involved
334
in the ECM-receptor interaction, focal adhesion, cancer, p53 signaling, complement, coagulation and
335
TGF-beta signaling pathways. Of the 433 proteins identified, 58 proteins were dysregulated secreted
336
proteins, and they were mainly ECM proteins, growth factor binding proteins, proteases, and protease
337
inhibitors, as well as proteins involved in catalysis, structural and receptor binding functions. Since this
338
in vitro study was conducted using cultured cells, we could not exclude the possibility that some findings
339
might not be recapitulated in actual tissues. Furthermore, sample normalization using the total protein
340
concentration might introduce some artificial effects on quantification. An increase concentration of one
341
protein would unlikely accompany by a decrease concentration of another in physiological condition.
342
Secreted proteins are attractive drug targets due to their accessibility. It is, therefore, imperative to
343
examine the secreted proteome further for translational medicine of complex diseases, such as
344
pterygium. Emerging label-free and targeted quantitative proteomics strategies would be more
345
appropriate for the future studies.
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346 Acknowledgement
348
This research is supported by the Singapore National Research Foundation under its
349
NMRC/CSA/045/2012, NMRC/BNIG/1075/2012 and administered by the Singapore Ministry of Health’s
350
National Medical Research Council. The funders had no role in study design, data collection, and
351
analysis, decision to publish, or preparation of the manuscript. Aihua Hou would like to thank Anuradha
352
Veerappan for proofreading of the original manuscript.
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353 354
Author contributions
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AH and MQT cultured primary pterygium and conjunctival fibroblast cells, collected conditioned medium
356
and concentrated secreted proteins. KPL designed and conducted the iTRAQ experiments, prepared the
357
samples and performed the data analysis. YPL conceived the experimental designed the study. AH, KPL
358
and LT wrote the manuscript.
359
An, M.X., Wu, K.L., Lin, S.C., 2011. Detection and comparison of matrix metalloproteinase in primary and
363
recurrent pterygium fibroblasts. Int J Ophthalmol 4, 353-356.
364
Argraves, W.S., Greene, L.M., Cooley, M.A., Gallagher, W.M., 2003. Fibulins: physiological and disease
365
perspectives. EMBO Rep 4, 1127-1131.
366
Barker, T.H., 2011. The role of ECM proteins and protein fragments in guiding cell behavior in
367
regenerative medicine. Biomaterials 32, 4211-4214.
368
Bendtsen, J.D., Jensen, L.J., Blom, N., Von Heijne, G., Brunak, S., 2004a. Feature-based prediction of non-
369
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Bendtsen, J.D., Kiemer, L., Fausboll, A., Brunak, S., 2005. Non-classical protein secretion in bacteria. BMC
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Microbiol 5, 58.
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Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004b. Improved prediction of signal peptides:
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Blaber, S.P., Webster, R.A., Hill, C.J., Breen, E.J., Kuah, D., Vesey, G., Herbert, B.R., 2012. Analysis of in
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Blum, H.F., 1959. Carcinogenesis by Ultraviolet Light. Princeton University Press.
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Table 1. Pathway analysis of the 135 significantly different proteins between pterygium and conjunctiva.
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% PValue 15.67 0.000 14.93 0.000 6.72 0.065 5.97 0.022 5.22 0.001 4.48 0.001
6 5
4.48 3.73
0.002 0.067
4 3 19 5 3 19 11 11 11
2.99 2.24 14.18 3.73 2.24 14.18 8.21 8.21 8.21
0.039 0.042 0.000 0.000 0.037 0.000 0.000 0.000 0.032
10 7
7.46 5.22
0.000 0.007
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REACTOME_PATHWAY REACTOME_PATHWAY
Count 21 20 9 8 7 6
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KEGG_PATHWAY KEGG_PATHWAY PANTHER_PATHWAY PANTHER_PATHWAY PANTHER_PATHWAY REACTOME_PATHWAY REACTOME_PATHWAY REACTOME_PATHWAY REACTOME_PATHWAY
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KEGG_PATHWAY KEGG_PATHWAY
Term hsa04510:Focal adhesion hsa04512:ECM-receptor interaction hsa05200:Pathways in cancer hsa04810:Regulation of actin cytoskeleton hsa05222:Small cell lung cancer hsa00010:Glycolysis / Gluconeogenesis hsa04610:Complement and coagulation cascades hsa04142:Lysosome hsa05130:Pathogenic Escherichia coli infection hsa00030:Pentose phosphate pathway P00034:Integrin signaling pathway P00024:Glycolysis P00050:Plasminogen activating cascade REACT_604:Hemostasis REACT_18266:Axon guidance REACT_16888:Signaling by PDGF REACT_15380:Diabetes pathways REACT_13552:Integrin cell surface interactions REACT_474:Metabolism of carbohydrates
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Category KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY
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Table 2. Dysregulated proteins in conditioned media of pterygium fibroblasts.1 Accession Gene Symbol SP2
RI PT
yes
202
1.406
0.000
13.183
0.000
9.204
0.000
COL12A1
yes
81
2.291
0.000
2.312
0.000
4.130
0.000
P12109
COL6A1
yes
85
2.312
0.000
9.727
0.000
6.546
0.000
P07942
LAMB1
yes
45
1.355
0.000
4.920
0.002
5.495
0.002
Q16363
LAMA4
yes
41
P98160
HSPG2
yes
40
P09871
C1S
yes
31
P00736
C1R
yes
P12110
COL6A2
yes
Q15113
PCOLCE
yes
Q9UI42
CPA4
yes
Q4LDE5
SVEP1
P08294
SOD3
P01344
IGF2
P02545
LMNA
M AN U
SC
COL6A3
0.004
1.406
0.002
15.276
0.000
3.373
0.001
1.871
0.000
1.644
0.054
3.221
0.002
2.805
0.000
8.166
0.000
39
3.467
0.000
1.570
0.001
3.192
0.103
50
1.754
0.002
7.586
0.000
8.241
0.000
26
1.722
0.001
1.459
0.002
28.576
0.000
22
2.884
0.234
1.419
0.021
32.211
0.000
yes
18
1.472
0.541
1.706
0.001
4.246
0.002
yes
7
6.368
0.004
2.466
0.031
1.675
0.090
yes
5
3.311
0.006
12.023
0.000
3.837
0.325
Ex3
20
2.228
0.000
1.271
0.070
2.355
0.000
EP
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1.380
AC C
UpP12111 regulated Q99715
Peptides 114:113 p-value 116:115 p-value 118:117 p-value (95%) 114:113 116:115 118:117
2
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FBLN1
yes
41
8.872
0.001
0.904
0.318
10.186
0.001
P24043
LAMA2
yes
14
2.754
0.002
1.096
0.708
7.244
0.003
P51884
LUM
non-classical 16
1.675
0.001
25.119
0.007
1.236
0.019
P37802
TAGLN2
non-classical 18
1.660
0.040
0.847
0.361
2.377
0.004
Q14204
DYNC1H1
ex
3
1.570
0.001
1.675
0.013
1.127
0.004
P08123
COL1A2
yes
531
1.706
0.000
0.258
0.000
1.600
0.000
P08670
VIM
non-classical 99
1.585
0.032
0.445
0.000
2.992
0.000
P18206
VCL
ex
35
1.380
0.045
0.501
0.012
2.051
0.000
Q13813
SPTAN1
ex
15
1.660
0.033
1.738
0.009
0.698
0.001
P36955
SERPINF1
yes
21
5.445
0.000
0.597
0.133
5.058
0.001
P05997
COL5A2
yes
P08572
COL4A2
yes
P35442
THBS2
yes
Q01995
TAGLN
P04264
KRT1
P51888
PRELP
DownP02751 regulated P02452
TE D
M AN U
SC
RI PT
P23142
3.631
0.004
0.122
0.004
1.959
0.005
20
1.977
0.002
0.196
0.025
2.070
0.002
14
2.270
0.007
4.246
0.001
0.441
0.006
non-classical 24
2.443
0.004
0.138
0.000
2.421
0.000
AC C
EP
26
ex
8
3.733
0.045
8.395
0.001
0.015
0.009
yes
7
3.162
0.016
17.378
0.001
0.497
0.552
FN1
yes
349
0.128
0.000
0.146
0.000
0.213
0.000
COL1A1
yes
426
0.207
0.088
0.466
0.020
0.520
0.002 3
ACCEPTED MANUSCRIPT
THBS1
yes
47
0.270
0.000
0.692
0.040
0.118
0.000
O00391
QSOX1
yes
37
0.229
0.000
0.249
0.000
0.466
0.000
P05121
SERPINE1
yes
57
0.221
0.000
0.041
0.000
0.275
0.000
Q12805
EFEMP1
yes
54
0.095
0.000
0.597
0.077
0.094
0.001
P09486
SPARC
yes
84
0.113
0.014
0.196
0.001
0.625
0.776
Q15582
TGFBI
yes
43
0.391
0.000
0.145
0.000
0.597
0.082
P01033
TIMP1
yes
40
0.229
0.119
0.316
0.022
0.036
0.000
P00338
LDHA
ex
23
0.163
0.000
0.515
0.012
0.240
0.000
Q14767
LTBP2
yes
19
0.421
0.000
0.409
0.000
0.305
0.000
Q05682
CALD1
Un4
16
0.631
0.430
0.402
0.000
0.470
0.010
P17936
IGFBP3
yes
Q08629
SPOCK1
yes
P07858
CTSB
yes
Q7Z7G0
ABI3BP
yes
P13611
VCAN
Q14766
LTBP1
P55268 Q76M96
TE D
M AN U
SC
RI PT
P07996
0.291
0.004
0.631
0.260
0.021
0.002
13
0.445
0.033
0.483
0.026
0.091
0.007
6
0.479
0.043
0.649
0.275
0.296
0.007
6
0.409
0.031
0.366
0.076
0.029
0.002
yes
8
0.213
0.011
0.064
0.083
0.018
0.030
yes
4
0.120
0.039
0.108
0.056
0.187
0.058
LAMB2
yes
40
0.912
0.502
0.679
0.000
0.029
0.000
CCDC80
yes
27
1.202
0.419
0.319
0.000
0.045
0.000
AC C
EP
16
4
ACCEPTED MANUSCRIPT
SERPINE2
yes
38
0.429
0.001
0.991
0.842
0.090
0.000
Q9UBP4
DKK3
yes
17
1.148
0.436
0.087
0.011
0.247
0.002
P00558
PGK1
ex
10
0.328
0.020
0.254
0.014
1.057
0.432
P27816
MAP4
un
4
0.752
0.015
0.802
0.137
0.319
0.004
P35555
FBN1
yes
47
0.283
0.000
0.773
0.199
0.215
0.000
P03956
MMP1
yes
26
0.511
0.000
1.854
0.008
0.016
0.000
P24592
IGFBP6
yes
11
0.299
0.038
0.366
0.030
1.343
0.602
M AN U
SC
RI PT
P07093
at least in 2 out of 3 pairs of samples, Fold changes by iTRAQ Analysis
2
signaling peptides;
3
exosome;
4
unknown.
AC C
EP
TE D
1
5
ACCEPTED MANUSCRIPT
Table 3. Different pathways of which the dysregulated secreted proteins were involved in. Pathways1
Gene lists
RI PT
collagen, type I, alpha 1 collagen, type I, alpha 2 collagen, type IV, alpha 2
collagen, type VI, alpha 1
M AN U
collagen, type VI, alpha 2
SC
collagen, type V, alpha 2
collagen, type VI, alpha 3 fibronectin 1
ECM-receptor interaction
heparan sulfate proteoglycan 2 laminin, alpha 2
TE D
laminin, alpha 4 laminin, beta 1
laminin, beta 2 (laminin S)
EP
thrombospondin 1 thrombospondin 2
AC C
Focal adhesion
The same list as “ECM-receptor interaction” collagen, type IV, alpha 2
fibronectin 1 laminin, alpha 2
Pathways in cancer
laminin, alpha 4 laminin, beta 1 laminin, beta 2 (laminin S)
6
ACCEPTED MANUSCRIPT
matrix metallopeptidase 1 insulin-like growth factor binding protein 3 serpin peptidase inhibitor, clade E, member 1
p53 signaling pathway
RI PT
thrombospondin 1 complement component 1, r subcomponent Complement and coagulation cascades
complement component 1, s subcomponent
SC
serpin peptidase inhibitor, clade E, member 1
latent transforming growth factor beta binding protein 1 thrombospondin 1
M AN U
TGF-beta signaling pathway
thrombospondin 2
EP
TE D
DAVID, pathways, KEGG_PATHWAY were chosen.
AC C
1
7
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figures and Legends
Figure 1. Pterygium secretome experimental design.
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
Figure 2. (A) Pie chart showing the types of extracellular proteins identified by mass spectrometry. (B) Pie chart showing up- or down-regulated proteins in the conditioned media of pterygium fibroblasts.
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Figure 3. Bar charts showing biological function analysis results of the up- or down-regulated proteins by PANTHER.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 4. A. Protein expression of Fibulin-1 and SERPINE1 in conditioned media of pterygium and conjunctival fibroblast cells was determined by immunoblotting with antibodies specific for Fibulin-1 and SERPINE1. B. Densitometry measurement of the bands in A.
ACCEPTED MANUSCRIPT
Highlights
RI PT
Secreted proteins collected from paired pterygium and conjunctival fibroblasts were identified and quantified by iTRAQ-based tandem mass spectrometry. 433 proteins were identified, of which 31 were up-regulated and 27 were down-regulated in conditioned media of pterygium fibroblast compared to control. Molecular function analysis suggested that these proteins belonged to catalytic proteins, structural molecules or were involved with receptor activities and protein binding.
AC C
EP
TE D
M AN U
SC
Pathway analysis suggested that these proteins were involved in ECM-receptor interaction, focal adhesion, cancer, p53 signaling, complement, coagulation, and TGF-beta signaling pathways.