Differentially expressed proteins underlying childhood cortical dysplasia with epilepsy identified by iTRAQ proteomic profiling

Patients and tissue preparation

The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (2013–026), and the study was conducted according to the principle expressed in the Declaration of Helsinki. Written consents were obtained from patients and/or their legal guardians. No author but S. L. and H. Y. had access to information that could identify individual participants during data collection, while no author had access to such information after data collection.

All the included CCD patients were recruited from 2013 August to 2014 December, and were preoperatively assessed with detailed history, neurological examination, neuropsychological test, ictal and interictal electroencephalography and MRI together with intraoperative electrocorticography for diagnosis and localization of lesions. After surgery, brain tissues from CCD patients were diagnosed by neuropathologists according to consensus of International League Against Epilepsy [10]. All included TIH patients underwent surgery had no history of neurological diseases, and the resected brain tissues were histologically normal. The resected tissues were immediately immersed into liquid nitrogen and strored at -80°C. Brain tissues of sex- and age-matched 8 CCD patients and 8 controls were selected for iTRAQ, brain tissues from the rest 15 CCD patients and 15 controls were used for qPCR, immunoblotting and immunohistochemistry.

Sample preparation and iTRAQ reagents labeling

Total proteins were extracted with iTRAQ lysis buffer. The concentration of proteins was measured using 2-D Quant Kit (Amersham Biosciences, Uppsala, Sweden). Equal amount of proteins from each group were mixed. The pooled samples were subjected to iTRAQ labeling according to the iTRAQ kit protocol (Applied Biosystems, Framingham, MA, USA). Briefly, 2 μl reducing reagent was added to 200 μg protein and centrifuged, then it was incubated at 37°C for 1 hour. 1 μl of Cysteine-Blocking Reagent was added for cysteine blocking. Each protein sample was digested into peptide with 4 μg Trypsin overnight at 37°C. iTRAQ reagents were dissolved in isopropanol, and then mixed with the corresponding sample followed by incubation at room temperature for 3 hours. Samples from CCD patients were labeled with 118 tag and 121 tag, and samples from controls were labeled with 117 tag and 119 tag. All the iTRAQ reagent-labeled samples were then combined. [11, 12]

Peptide fractionation with Isoelectric Focusing (IEF)

The labeled peptides samples were fractionated by IEF on immobilized pH gradient as described previously [13–15]. Briefly, the labeled peptides were dissolved in urea and Pharmalyte solution, applied to IPG strips (pH 3–10), and then focused with an IPGphor system (GE Healthcare Life Sciences Amersham Biosciences, Pittsburg, PA, USA) at 68 kVh. The IPG strips were cut into 36 pieces (0.5cm per piece). Peptides in each pieces was extracted with 0.1% formic acid and 2% acetonitrile and lyophilized and desalted with a C18 Discovery DSC-18 SPE column (Sigma-Aldrich). The desalted peptides were lyophilized again and stored at -20°C for mass spectrometry analysis.

Mass spectrometry and gene oncology analysis

Mass spectrometry was performed with liquid chromatography coupled inline to a QStar mass spectrometer (Applied Biosystems, Framingham. MA, USA). Desalted peptides were reconstituted in a solution containing 0.1% formic acid and 2% acetonitrile, half of which was delivered into a trap column by an online capillary liquid chromatography system (Dionex Ultimate 3000, Amsterdam, The Netherlands). The peptide mixture were automatically separated on a C18-PepMap column (ThermoFisher Dionnex, Sunnyvale, CA, USA) at 0.3 μl/min. The eluent was analysed by OStar Elite Hybrid ESI Quadrupole time-of-flight tandem mass spectrometer (Applied Biosystems, Framingham. MA, USA) in an information-dependent acquisition mode. Mass spectrometer data acquisition was performed in the positive ion mode, with a selected mass range of 300–1800 m/z. A setting of 2 s was used as the total time for MS/MS events. The two charged peptides which were most abundant, with more than 20 counts, were selected for MS/MS and dynamically excluded for 30 s with ± 50 mDa mass tolerance.

Peptide identification and quantification was performed by ProteinPilot software (Applied Biosystems, Framingham, MA, USA). The search was performed using the International Protein Index (IPI) human database v3.87. Cysteine modification by MMTS was specified as a fixed modification [16].

For protein identification, a generally accepted standard which has been widely used in identifying and quantifying proteins with iTRAQ was taken [17–20]. The protein threshold was set to achieve 95% confidence, False discovery rate (FDR) statistics and 1.3-fold change cut-offs were used to classify the protein expressions as up-regulated (FDR<0.05 and iTRAQ fold-changes above 1.3) or down-regulated (FDR<0.05 iTRAQ and fold-changes below 0.77). For technical variation, while an analysis of repeated iTRAQ experiments established the technical variability to be not more than 30%. The gene oncology of each differentially expressed proteins was searched and classified using PANTHER classification system (www.pantherdb.org). 8 differentially expressed proteins which have not been reported associated with CCD, but may participate in CCD-related biological processes or have CCD-related molecular functions according to previous literature were selected for further validation.

Real-time qPCR analysis

Total RNA was extracted using Trizol (Thermofisher, Waltham, USA). Extracted RNA was reverse transcribed into cDNA by A3500 Reverse Transcription System (Promega, Madison, WI, USA). qPCR was performed using TaqMan GeneExpression Kit in ABI 7900HT system. The sequences of primers (OriGene Technologies, Inc. Rockville, USA) were NDRG1 (HP209104), FSCN1 (HP206673), FABP3 (HP207465), DPYSL5 (HP213501), CRMP1 (HP232913), PRDX6 (HP208150), PSAP (HP231407), MAP4 (HP206072), and β-Actin (HP204660). The mRNA expression level were analyzed using ΔΔCt method.

Immunoblotting analysis

Total proteins were extracted with RIPA Lysis Buffer and the concentrations were determined with BCA Kit (Beyotime, Haimen, China). Protein samples were loaded to 10% SDS-PAGE gel for electrophoresis and transferred to PVDF membranes. The membranes were incubated in 0.4% gelatin for 1 hour at room temperature and then incubated in primary antibodies (CRMP1, DPYSL5, FSCN1, NDRG1, PRDX6) (1:1000–1:10000 dilution, Abcam, Cambridge, UK) at 4°C overnight. After washed with TBST buffer, the membranes were incubated in HRP-conjugated secondary antibody (1:5000 dilution) for 1h at room temperature. The protein bands were visualized using ECL detection system (Millipore, Germany) and analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Immunohistochemistry

Brain tissues from CCD patients and controls were fixed with 4% paraformaldehyde overnight at 4°C, then routinely embedded in paraffin and sectioned. After dewaxing and rehydration, the sections were boiled in citrate buffer (pH 6.0) in microwave oven for 20 min for antigen retrieval. Endogenous peroxidase activity was quenched by treatment with 3% H₂O₂ for 10 min. The sections were blocked with goat serum for 30 min and incubated at 4oC overnight with primary antibodies (CRMP1 1:250, DPYSL5 1:100 and FSCN1 1:250). Then sections were incubated in goat-anti-rabbit secondary antibodies (30 min, 37°C) and visualized using DAB (ZSGB-Bio, Beijing, China). After counterstain with hematoxylin and dehydrated, sections were evaluated under microscope. A semi-quantitative score was applied to the images obtained from the microscope inspection. The staining intensity ranged from 1 to 3 and the percentage of positive cells was measured manually in a range from 0 to 100%. Staining intensity (1–3) was multiplied by the percentage of positive cells (0–100) to obtain a final score ranging from 0 to 300 [21]. This method was repeated three times for the immunohistochemistry of each antibody in the brain tissue samples.

Statistical analysis

Data was expressed as mean±SD, FDR statistics was used to identified differentially expressed proteins, FDR<0.05 was considered statistically significant. Intergroup differences in immunoblotting and immunohistochemistry between the CCD group and the control group were analyzed using t test or rank sum test. p<0.05 was considered statistically significant.

Demographics and clinical characters of patients

23 CCD patients (11 female, age 6.96±3.70, disease course 2.76±1.61 years) and 23 TIH patients (8 Female, age 7.22±3.10 years) who underwent surgery were included in this study. (Table 1)

Differentially expressed proteins revealed by iTRAQ analysis

Brain tissues of 16 randomly selected patients (n = 8 for each group) were analyzed with iTRAQ. In total, 3440 proteins were found with 95% confidence, among which 153 proteins were differentially expressed (FDR<0.05), including 64 up-regulated and 89 down-regulated proteins (Tables 2 & 3).

Using PANTHER classification system, the 153 proteins were divided into 9 functional categories including catalytic activity (38.0%), binding (26.3%), molecule-structuring activity (14.0%), transporter activity (8.8%), and enzyme regulation activity (5.3%) (Fig 1). 8 differentially expressed proteins, including FSCN1, CRMP1, NDRG1, DPYSL5, MAP4, FABP3, PRDX6 and PSAP were selected for further validation. The gene oncology terms of these 8 proteins were shown in S1 Table.

qPCR in children having CCD with epilepsy

The levels of mRNA expression for fascin actin-bundling protein 1 (FSCN1), collapsin response mediator protein 1 (CRMP1), N-myc downstream regulated 1 (NDRG1), dihydropyrimidinase-related protein 5 (DPYSL5), peroxiredoxin 6 (PRDX6), prosaposin (PSAP), microtubule associated protein 4 (MAP4), and fatty acid binding protein 3 (FABP3) are presented in Fig 2. The expression of FSCN1, CRMP1, NDRG1, DPYSL5, MAP4, and FABP3 were found to be up-regulated in the CCD patients (Relative mRNA expression: CRMP1, 2.21±0.12; NDRG1, 3.61±0.11; DPYSL5, 2.73±0.12; MAP4, 3.17±0.08; FAPB3, 2.88±0.06. p<0.05 for each mRNA expression compared to controls), and the expression of PRDX6 and PSAP were down-regulated (Relative mRNA expression: PRDX6, 0.35±0.14; PSAP, 0.24±0.06. p<0.05 for both mRNA expression compared to controls) compared to controls.

Immunoblotting

CRMP1, DPYSL5, FSCN1, NDRG1, PRDX6 were further validated with immunoblotting. In CCD patients, the protein levels of CRMP1 (CCD group: 1.31±0.35; Controls: 0.41±0.15. p<0.05), DPYSL5 (CCD group: 1.04±0.07; Controls: 0.11±0.03. p<0.05), FSCN1 (CCD group: 1.89±0.18; Controls: 1.02±0.17. p<0.05) and NDRG1 (CCD group: 0.38±0.04; Controls: 0.04±0.01. p<0.05) were increased, while the protein level of PRDX6 (CCD group: 0.34±0.06; Controls: 2.00±0.37. p<0.05) was decreased compared to controls (Fig 3).

Immunohistochemistry

The expression of DPYSL5, CRMP1 and FSCN1 were further measured by IHC. There were increased immunoreactivity of CRMP1 (Scores: CCD group: 95.50±25.52; Controls: 15.76±5.28. p<0.05) (Fig 4), DPYSL5 (Scores: CCD group: 90.93±13.15; Controls: 29.33±5.21. p<0.05) (Fig 4), and FSCN1 (Scores: CCD group: 126.53±30.70; Controls: 15.06±3.45. p<0.05) (Fig 4) in the CDD group compared to controls.