1.1 Material
Seventeen minority children (Zhuang, 11 cases; Yao, 6 cases) diagnosed with epilepsy from November 2017 to February 2018 in Nanning Fifth People's Hospital were selected as the experimental group. Nine healthy minority children (Zhuang, 2 cases; Yao, 7 cases) with matched genders and ages were selected as the control group. There were 13 males and 4 females in the experimental group, with an average age of 9.41 ± 0.63 years, and 6 males and 3 females in the control group, with an average age of 11.11 ± 0.27 years. There was no significant difference in age, gender or ethnicity between the two groups (P > 0.05), and the samples were comparable (see Appendix A; A. Histogram of age distribution in the two groups. B. Bar graph of gender distribution in the two groups. C. Bar graph of ethnic distribution in both groups).
1.2 Diagnostic Criteria
The Clinical Guidelines for Epilepsy released by the International Anti-Epilepsy Alliance
(ILAE) in 2014 were used as a basis for diagnosis.
1.3 Inclusion Criteria
(1) Disease manifestation was consistent with the abovementioned Clinical Guidelines for
Epilepsy.
(2) The patients were 2 to 14 years old and had experiences typical seizures more than 2 times. There were no positive signs in the nervous system examination and no abnormalities in the head MRI. The patients did not receive any prior anti-epilepsy treatment. Informed consent was reviewed and signed by parents or legal guardians.
1.4 Exclusion Criteria
(1) Patients presented with other brain diseases or major organ dysfunction.
(2) Patients presented with mental retardation, febrile seizures, non-epileptic seizures and abnormal head MRI.
1.5 Reagents and Equipment
The following instruments were used: a high-speed centrifuge (Eppendorf); a C18 desalting column: Strata X C18 (Phenomenex); an e2695/2998 high-performance liquid chromatography (HPLC) system (Waters); a Bridge Peptide BEH C18 (130 Å, 3.5 µm, 4.6*250 mm) high-pH C18 RP column (Waters); a NanoLC 1000 nanoscale HPLC system (Thermo); and a Q Exactive (Thermo, Massachusetts, USA) for combined mass spectrometry. Urea, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), etc., were used for protein extraction. A Bradford assay kit (Biyuntian) was used for protein quantification. DTT, iodoacetamide (IAM), trypsin and trypsin spin columns were used for enzymatic labelling. The iTRAQ8plex® Reagent Kit (AB Sciex, Boston, USA) was used. Unless otherwise specified, the biochemical reagents used in this study were purchased from Sigma.
1.6 Method
1.6.1 Collection and Treatment of Serum Specimens
Three-millilitre venous blood specimens were collected from children on an empty stomach in blood collection tubes containing heparin as an anticoagulant and centrifuged at 4°C (1,500 r/min, centrifuge radius 19.5 cm, 10 min). Two to three serum samples were randomly pooled as a biological replicate, and there were 10 replicates in the experimental group and 5 replicates in the control group. Serum samples were depleted of high-abundance proteins using the ProteoPrep® Immunoaffinity Albumin and IgG Depletion Kit (Sigma-Aldrich, No. PROTIA-1KT). The protein concentration was determined by the Bradford method.
1.6.2 Enzymatic Digestion and Peptide Labelling
Samples (100 µg) were aliquoted based on the quantitative and SDS-PAGE results. All samples were adjusted to equal volumes. The protein disulphide bonds were reduced by adding DTT at a final concentration of 10 mM and incubating for 45 min at 37°C. Alkyl groups were protected by incubation at room temperature for 30 min with IAM at a final concentration of 25 mM. Urea was diluted with 100 mM triethylammonium bicarbonate (TEAB) to a final concentration of less than 2 M for enzymatic hydrolysis. Trypsin was added at a mass ratio of 1:50, and digestion was performed overnight at 37°C. Then, trypsin was added again at a mass ratio of 1:100, and the second hydrolysis was performed at 37°C for 4 h. The enzymatically obtained peptides were desalted using Strata X C18. After vacuum drying, iTRAQ8plex was used for labelling. Briefly, 50 mM TEAB was used to reconstitute and desalt peptides. Forty-one microlitres of isopropanol was used to reconstitute 0.8 mg of iTRAQ8plex reagent, which was then mixed with 100 µg of peptides to incubate for 2 h at room temperature. In the iTRAQ8plex labelling experiment, 10 disease group samples (from a mixture of 17 randomly paired samples and 3 independent samples) and 5 healthy samples (from a mixture of 9 randomly paired samples and a single independent sample) were included. A total of 3 groups of samples were included in the iTRAQ8plex labelling experiment, the first two groups among which contained 2 healthy samples and 4 disease group samples, using 6 of the 8 reagents. Two healthy samples were labelled with 113 and 114, and 4 disease group samples were labelled with 115, 116, 117, and 118. Group 3 contained one healthy sample (113) and two disease group samples (115, 117). Finally, the peptides were vacuum dried by a benchtop vacuum concentrator.
1.6.3 Peptide Mass Spectrometry Identification and Database Analysis
The mixed labelled peptide fragments were preseparated into 16 fractions by HPLC on a C18 column. The 16 components were dissolved in 20 µL of NanoLC A solution (98% H2O, 2% acetonitrile, 0.1% FA). Two micrograms of each sample was loaded onto the loading column and eluted by the gradient column into a Q Exactive instrument for combined mass spectrometry analysis. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS. Full-scan MS spectra (from m/z 300 to 1800) were acquired in the Orbitrap with a resolution of 70,000. Ion fragments were detected in the Orbitrap at a resolution of 17,500, and the 15 precursors with the most intense signals were selected for subsequent decision tree-based ion trap HCD fragmentation at a collision energy of 32% in the MS survey scan with 10.0 s dynamic exclusion. The Nano-HPLC conditions were as follows: peptides were reconstituted in solution A (0.1% formic acid, 2% acetonitrile, 98% H2O) and directly loaded onto a reversed-phase precolumn (Acclaim PepMap®100 C18, 3 µm, 100 Å, 75 µm× 2 cm) at 5 µL/min in 100% solvent A (0.1 M acetic acid in water). Next, peptides eluted from the trap column were loaded onto a reversed-phase analytical column (Acclaim PepMap® RSLC C18, 2 µm, 100 Å, 50 µm × 15 cm). The main gradient comprised an increase from 12–35% solvent B (0.1% FA in 98% ACN) over 35 min during a 55 min injection at a constant flow rate of 300 nL/min in an EASY-nLC 1000 system. The resulting raw MS/MS data were searched against the reviewed Swiss-Prot Homo sapiens database using Sequest software integration in Proteome Discoverer (version 1.3, Thermo Scientific). Trypsin was chosen as the enzyme, and two missed cleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, and oxidation (M) and acetylation at the N-terminus were set as variable modifications. The searches were performed using a peptide mass tolerance of 20 ppm and a product ion tolerance of 0.05 Da, resulting in a 1% false discovery rate (FDR).
1.6.4 Bioinformatics Analysis
Then, the proteins were classified by GO annotation based on three categories: biological process (BP), cellular component (CC) and molecular function (MF). The GO annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Pathway analysis was performed by using the Kyoto Gene and Genome Encyclopedia (KEGG) database (https://www.kegg.jp/). First, the KEGG Automatic Annotation Server (KAAS) online service was used to annotate the KEGG pathway of the proteins. Then, the KEGG Mapper online tool was used to map the annotation results to the KEGG pathway database, and the InterProScan-based protein sequence alignment method and InterPro domain database were used to annotate the protein domains. The protein-protein interaction (PPI) network was analysed by using the STRING database with a confidence level of 0.4 (maximum of 1). Critical networks were generated by Cytoscape 3.7.1. The key network was generated by using the cytoHubber application in Cytoscape 3.7.1 (where the circle size indicates degree and the circle colour indicates fold change), and the enriched network nodes were annotated by using the KEGG pathway database.
1.6.5 Statistical Analysis
The P-values of the samples were calculated using the T-test function in R. A difference between two groups of differentially expressed proteins was considered to be significant at a P < 0.05 and a differential expression ratio > 1.3. Fisher’s exact test was used for identification of differentially expressed proteins. Enrichment analysis was performed using Fisher's exact test, and P < 0.05 was considered to indicate significant enrichment of a particular GO function.