iTRAQ-based proteomic profiling reveals different protein expression between normal skin and hypertrophic scar tissue

Background A hypertrophic scar is a unique fibrotic disease that only exists in humans. Despite advances in burn care and rehabilitation, as well as progress in the management during these decades, the hypertrophic scar remains hard to cure following surgical methods and drugs for treatment. In this study, we are looking forward to finding the multitude of possible traumatic mechanisms and the underlying molecular signal ways in the formation of the hypertrophic scar. Methods We used isobaric tags for relative and absolute quantitation (iTRAQ) labeling technology, followed by high-throughput 2D LC-MS/MS, to determine relative quantitative differential proteins between the hypertrophic scar and normal skin tissue. Results A total of 3166 proteins were identified with a high confidence (≥95 % confidence). And, a total of 89 proteins were identified as the differential proteins between the hypertrophic scar and normal skin, among which 41 proteins were up-regulated and 48 proteins were down-regulated in the hypertrophic scar. GO-Analysis indicated the up-regulated proteins were involved in extracellular matrix, whereas the down-regulated proteins were involved in dynamic junction and structural molecule activity. Conclusions In our study, we demonstrate 89 proteins present differently in the hypertrophic scar compared to normal skin by iTRAQ technology, which might indicate the pathologic process of hypertrophic scar formation and guide us to propose new strategies against the hypertrophic scar.


Background
A hypertrophic scar, a unique fibrotic disease in humans as no animals are known to form these lesions, presents as erythematous, firm, elevated plaques that remain confined to the area damaged by the initial injury [1]. Although it does not pose a health risk, some scars may be associated with pruritus, pain, disfigurement, disfunction, and psychological distress [2,3]. Despite advances in burn care and rehabilitation, as well as progress in the management during these decades, the pathologic scar remains hard to cure following surgical methods and drugs for treatment [4,5]. On this background, we are looking forward to finding the multitude of possible traumatic mechanisms and the underlying molecular signal ways in the formation of the hypertrophic scar.
In our previous studies, we have compared the global gene profiling from the normal skin and hypertrophic scar samples via cDNA microarray analysis [6]. The altered genes were related to proto-oncogenes, apoptosis, immune regulatory genes, cytoskeletal element, metabolism, and so forth. Besides the genomic approach, we further compared the protein profiles with a proteomic approach in this study. The isobaric tags for relative and absolute quantitation (iTRAQ) is a shotgun-based technique which allows the concurrent identification and relative quantification of hundreds of proteins in up to different biological samples in a single experiment [7]. With the iTRAQ approach, a large-scale evaluation of differential protein expression in the formation of the hypertrophic scar may be valuable for finding scarrelated proteins or the potential target to control the hypertrophic scar.
In this article, we revealed the differential proteomics between the hypertrophic scar and normal skin tissues. From three patients, 3166 proteins were screened by iTRAQ. Forty-one up-regulated proteins were related to extracellular matrix, and 48 down-regulated proteins were involved in dynamic junction and structural molecule activity.

Tissue procurement and patient characteristics
The patient informed consent forms along with tissue procurement procedures were approved by the Ethic Committee of Southwest Hospital, Chongqing, China. All the hypertrophic scar patients were selected according to the Vancouver Scar Scale (VSS) ranging from a score of 10 to 13. The hypertrophic scar tissues and the normal skin tissues were obtained from the same patients who underwent orthopedic surgery at the Institute of Burn Research of Southwest Hospital. The tissues were frozen in liquid nitrogen immediately after surgical removal and stored at −80°C till sample preparation.

Reagents and chemicals
The chemical reagents acetonitrile, ethanol, methanol, acetone, ammonium formate (high-performance liquid chromatography (HPLC) grade), and trifluoroacetic acid (TFA) were obtained from Sigma Corporation (Sigma, USA) and Fisher Science Corporation (Thermo, USA). The ultrapure-grade water, utilized for the HPLC and subsequent tandem mass spectrometry (MS/MS) analysis procedures, was generated from the MilliQ (Millipore, USA)-type water. All iTRAQ reagents and buffers were

Protein extraction
The tissues were minced to pieces of approximately 2 mm in size. The pieces of tissues were grinded with a mortar and pestle in liquid nitrogen. The soluble protein was extracted according to the protocol in the Partial Mammalian Proteome Extraction Kit (Calbiochem, USA). A volume of 1 ml of extraction reagent 1, 5 μl of protease inhibitor cocktail, and 500 μl glass beads were added immediately into 250 mg of the tissue powder. The sample was vortexed thoroughly for 1 min. After addition with a volume of 4 μl of Benzonase® Nuclease (Novagen, USA), the sample was incubated at 4°C for 30 min at 500 rpm. The solubilized sample was centrifuged at 15,000 rpm for 30 min at 4°C after ultrasonication. Then the precipitate was disintegrated with 1 ml detergent containing 500 μl 1 % (w/v) sodium dodecyl sulfate (SDS), 100 μl 1 % (v/v) NP-40, and 400 μl 10 M urea. Subsequently, the sample was incubated at 4°C for 30 min at 500 rpm. And, the protein of low solubility was extracted after the ultrasonication. The sample was centrifuged at 15,000 rpm for 30 min at 4°C, and the protein in the supernatant material was subjected to eight times the volume of ice-cold acetone precipitation overnight before re-suspending into 0.1 % (w/v) SDS, 0.1 % (v/v) NP-40, and 1 M urea. The soluble protein and the low soluble protein were quantified by the BCA protein assay kit (Pierce, USA) and stored in aliquots at −80°C until use.

In-solution digestion
The low soluble protein aliquot containing 250 μg of total protein in 250 μl was reduced by the addition of 25 μl of 100 mM dithiothreitol (DTT; final concentration is 10 mM) followed by incubation at 56°C for 1 h.
The  Darker nodes refer to the significant ontologies of the dataset. The size is proportional to the number of genes that participate in that molecular function 80 min, 500 nl/min) was used for peptide elution, followed by a 5-min wash with 80 % B. A QStar Elite QqTOF mass spectrometer (Applied Biosystems) was used in standard MS/MS data-dependent acquisition mode with a nano-electrospray ionization source. Survey MS spectra were collected (m/z 400 to 1500) for 1 s followed by three MS/MS measurements on the most intense parent ions (80 counts/s threshold, +2 to +4 charge state, and m/z 100 to 1500 mass range for MS/MS), using the manufacturer's "smart exit" and "iTRAQ" settings. The "smart exit" option is a standard feature of the QStar Elite instrument, which runs under control of Analyst QS 2.0 software. It allows termination of MS/MS spectrum collection when preset values of peak intensity (quality of the spectra) are reached. In this way, the instrument spends less time for MS/MS acquisition of abundant species and improves chances for detection of low-abundant ones. Spectral quality setting 5 (whole scale 1-20) was used throughout the experiments. Predefined "iTRAQ" settings adjust (increase) collisional energy to maximize the intensity of reporter ions (114, 115, 116, 117 Da) in MS/MS spectra. Individual Wiff files generated after QStar Elite analysis were converted to mascot generic file (MGF) format using Mascot.dll script in Analyst QS 2.0. Following this conversion, MGF files of individual fractions were combined into one using a merging script [9].

Database search and protein identification
The MS/MS data were analyzed using ProteinPilot software version 2.0.1 (Applied Biosystems/MDS Sciex, Concord, ON, Canada). The search parameters were complete modifications of Cys alkylation with IAA, and inbuilt iTRAQ analysis residue modifications settings were on. Those protein candidates with greater than or equal to 95 % identification confidence were used for further analysis [8]. The annotation of protein cellular localization and biological function was performed using PPI network (Ingenuity Systems, Inc., Redwood City, CA, USA).

Results
Overview of proteomic changes in the hypertrophic scar comparing normal skin A detailed analysis of the proteomic changes was performed using iTRAQ labeling. Reporters with masses of 114 and 115 were used to separately label biological replicates of normal skin, and reporters with masses of 116 and 117 were used for replicates of the hypertrophic scar. The four isobaric tag samples were mixed and analyzed by 2D-MS/MS. Relative protein levels were determined by Fig. 3 Cluster analysis of the scar-up-regulated PPI network by MCODE plugin in cytoscape software. We identified eight genes belonging to the highest cluster, i.e., the leader genes: COL1A1, COL1A2, COL3A1, COL5A1, COMP, FN1, THBS1, and TNC comparing peak intensities of the four reporter ions released from each pool of purified, labeled peptides. We identified 3166 distinct proteins. Eighty-nine proteins were identified with a high confidence to up-or down-regulate in the hypertrophic scar (Table 1).

Discussion
In this study, we use the iTRAQ method to screen the differential proteins potentially involved in the hypertrophic scar compared to normal skin. Using 2D-MS/ MS followed by GO-Analysis, we demonstrate 89 proteins with a high annotation confidence (≥95 %) present differently in the hypertrophic scar. Among the differential proteins, 41 proteins increased and 48 proteins decreased in the hypertrophic scar. Consistent with previous studies, the up-regulated proteins such as COL1A1, COL1A2, COL3A1, COL5A1, COMP, FN1, THBS1, and TNC were involved in extracellular matrix production, myofibroblast contractility, and response to mechanical stress [10][11][12][13]. Interestingly, most of the down-regulated proteins, including DSC3, DSP, EVPL, JUP, and PPL, were specific to the epidermis and involved in the cell junction [14][15][16][17][18].
Some of the differentially expressed candidates identified by iTRAQ have previously been associated with hypertrophic scar formation. Of the up-regulated proteins, increased COL1A1, COL1A2, COL3A1, FN1, and TNC expression have been reported in the hypertrophic scar. COMP plays a role as a matrix deposition promoter in the keloid (the special fibrotic skin disease) [19]. It has been reported that THBS1 may modulate keloid formation through up-regulation of the matrix plasminogen/plasmin system [20]. However, no data revealed the potential effects of COMP and THBS1 on the hypertrophic scar formation. Our work for the first time found COMP and THBS1 were up-regulated in the hypertrophic scar, which might be attributed to a new therapeutic target.

Conclusions
In summary, the iTRAQ analyses followed by the highthroughput 2D LC-MS/MS in our study for the first time screened protein expression of the hypertrophic scar and normal skin tissue on a large scale from the same patients. Some of the screened proteins in our study have been reported in previous researches. However, some of the upregulated proteins such as COMP and THBS1 and the down-regulated proteins could indicate that the pathologic process of hypertrophic scar formation which might guide us to propose new strategies against the hypertrophic scar.