Participants’ recruitment and eligibility criteria

Between October 2020 and December 2022, a total of 100 blood samples were collected at the Department of Neurology and Children’s Health Care, Shenzhen Children’s Hospital (Shenzhen, China). This cohort included 60 samples from patients with IESS (30 in the E F group and 30 in the IEF group) and 40 samples from the HC group. This study received ethical approval from the Ethics Committee of Shenzhen Children’s Hospital (Approval No. 202200802), and informed consent was obtained from the parents or legal guardians of each participant. The study adhered to the Declaration of Helsinki.

Participants were assessed by pediatric neurologists, and the diagnosis was based on the type of seizure, electroencephalographic results, and brain imaging findings. Inclusion criteria required participants to be under 2 years of age and to meet the diagnostic criteria for IESS as outlined by the International League Against Epilepsy (ILAE) in 2022 [1]. Exclusion criteria were applied to patients who were children experiencing seizures likely linked to acquired brain injuries, including those caused by head trauma, brain tumors, and central nervous system infections. Additionally, patients who had undergone allogeneic blood transfusions within the previous 2 months or had been diagnosed with hemolytic disease were excluded. HCs had to be under 2 years old and not have a personal or family history of epilepsy. All participants were of Chinese ethnicity. Following ACTH treatment, patients whose spasms were fully controlled or reduced by at least 80%, with resolution of hypsarrhythmia, were classified into the EF group, while those who did not meet these criteria were placed in the IEF group.

Preparation of proteins

A 5 mL venous blood sample was drawn into a tube containing ethylenediaminetetraacetic acid as an anticoagulant, which was then gently inverted 5 to 10 times to ensure thorough mixing of the blood. The samples were left to stand for 30 min to allow any potential hemolysis to subside, ensuring no vigorous shaking occurred. Following this, the samples underwent centrifugation at 2,000 g for a period of 10 min at 4 °C in a swinging bucket rotor. The supernatants were harvested, aliquoted, and immediately frozen in liquid nitrogen and subsequently preserved at −80 °C until required. Prior to analysis, the plasma was thawed in a water bath for 5 to 10 min. To prepare the samples for further processing, the supernatants were mixed with SDT buffer (4% SDS, 100 mM Tris-HCl, pH 7.6), and the resulting mixture was heated in a boiling water bath for 15 min. Following the heating step, the samples underwent centrifugation at 14,000 g for a duration of 20 min, and the resulting supernatants were carefully collected, and their protein concentrations were measured using the BCA Protein Assay kit, following the manufacturer’s guidelines (Bio-Rad, Hercules, CA, USA).

All samples were treated with DTT, adjusting the final concentration to 40 mM, and incubated at 600 rpm for 1.5 h at 37 °C. After incubation, the samples were allowed to return to room temperature, and in order to block reduced cysteine residues, IAA was added to a final concentration of 20 mM. The samples were then incubated in the dark for 30 min. The samples were transferred to 10 kDa Microcon units, washed three times with 100 µL of UA buffer, and rinsed twice using 100 µL of 25 mM NH₄HCO₃ buffer.

Trypsin was then added to each sample at a trypsin-to-protein weight ratio of 1:50. The samples were kept at 37 °C for 15 to 18 h, or overnight, to allow enzymatic digestion. The resulting peptide mixtures were collected as a filtrate. Subsequently, the peptides were subjected to desalting using C18 solid-phase extraction cartridges (Empore™ SPE Cartridges C18, standard density, 7 mm bed I.D., 3 mL volume; Sigma-Aldrich, MO, USA). The desalting process was followed by the concentration of the peptides through vacuum centrifugation. The concentrated peptides were dissolved in 40 µL of 0.1% (v/v) formic acid.

The peptide concentration was measured with UV absorbance at 280 nm. To facilitate DIA experiments, indexed retention time (iRT) calibration peptides were incorporated into the samples. After digestion, the pooled peptides were fractionated into 10 distinct fractions using the Thermo Scientific™ Pierce™ High pH Reversed-Phase Peptide Fractionation Kit. This process specifically aimed to target low-abundance components in plasma samples. Each fraction underwent desalting with C18 cartridges before reconstitution in 40 µL of a 0.1% (v/v) formic acid solution. For data-dependent acquisition (DDA) analysis, the reconstituted samples were supplemented with iRT peptides sourced from Biognosys (Schlieren, Switzerland) to ensure consistent retention time calibration across the samples.

MS assay for DDA and DIA

The separation process was carried out using an Easy-nLC 1200 liquid chromatography system (Thermo Scientific™) for DDA library generation. A C18 column (Thermo Scientific™, ES802, 1.9 μm, 75 μm × 20 cm) was used to perform the linear gradient separation. The mobile phase consisted of buffer B, which was prepared with 84% acetonitrile and 0.1% formic acid, and the flow rate was set at 300 nL/min. MS was performed in positive ion mode with a spray voltage of 1900 V. The full MS scan range was 350–1800 m/z, with a resolution of 60,000 at m/z 200. The automatic gain control (AGC) target was set to 1e6, and the maximum IT was 50 ms. A dynamic exclusion of 10.0 s was applied. Following each full MS–SIM scan, 20 ddMS2 scans were acquired based on an inclusion list. The isolation window was 1.5 m/z. MS2 scans were acquired at a resolution of 30,000 (at m/z 200), with an AGC target of 1e5, a maximum IT of 50 ms, and a normalized collision energy of 30 eV.

DIA proteomic analysis of each plasma sample was performed using a Q-Exactive HF-X mass spectrometer (Thermo Scientific™) coupled with an Easy-nLC 1200 chromatography system (Thermo Scientific™). Peptides were analyzed in DIA mode, with each cycle consisting of one full MS-SIM scan followed by 44 DIA acquisition windows covering a mass range of 350–1800 m/z. The full SIM scan was acquired at a resolution of 120,000 (at m/z 200), with an AGC target of 3e6, maximum IT of 30 ms, and operated in profile mode. DIA scans were acquired at a resolution of 30,000, with an AGC target of 3e6, maximum IT set to auto, using HCD with a normalized collision energy of 30 eV.

MS data analysis and DEP identification

Both DDA spectral library data and DIA data were analyzed using Spectronaut™ (version 14.4.200727.47784, Biognosys). Key software parameters included dynamic iRT for retention time prediction, enabled MS2-level interference correction, and cross-run normalization. All results were filtered using a Q-value cutoff of 0.01, corresponding to a false discovery rate (FDR) of less than 1%.

Protein identification was carried out with a rigorous confidence level of 99%, based on FDR ≤ 1%. The fold change (FC) comparing IESS patients to HCs was computed by averaging the expression levels of each protein. DEPs were selected based on stringent filtering criteria, with a log fold change (logFC) value greater than 1.5 (upregulated) or less than 0.67 (downregulated). Additionally, statistical significance was determined with a significance threshold of p < 0.05.

Soft clustering analysis

Soft clustering analysis was performed using the Mfuzz package, which assigns proteins to multiple clusters based on their clustering coefficients. This approach enabled the classification of DEPs through soft clustering techniques. The results from this analysis were then visualized using the R package ClusterGVis (version 0.1.1), providing a clear representation of the clustering outcomes.

Analysis of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment

To explore the significant biological pathways associated with the DEPs, the clusterProfiler package (version 4.10.0) was used to perform enrichment analyses for GO and KEGG pathways. The GO enrichment analysis covered three main categories: biological process (BP), cellular component (CC), and molecular function (MF). Both GO and KEGG pathway enrichment analyses were conducted using the Benjamini-Hochberg method to adjust for multiple testing. A significance threshold of p < 0.05 was applied to determine significance.

Protein−Protein Interaction (PPI) network

The PPI network for the DEPs was constructed using the STRING database (version 12.0, accessed on March 15, 2024, http://string-db.org/) [8]. A minimal confidence score of 0.4 was applied to the PPI interactions. A total of 24 proteins were selected for inclusion in the PPI network analysis based on the criteria of logFC greater than 1.5 or less than 0.67, with a significance threshold set at p < 0.05. The DEPs PPI networks were visualized and analyzed using the R packages ggraph (version 2.1.0) and igraph (version 1.5.1), with a maximum of 5 interactions allowed for each protein.

Receiver Operating Characteristic (ROC) curves

ROC curves were generated using the plot.roc function from the pROC R package (version 1.18.5). To evaluate the discriminatory ability of the candidate biomarkers, the auc function within the same package was used to calculate the areas under the curve (AUC). Additionally, ROC curve analysis was conducted using SPSS version 22.0 software (IBM, Armonk, NY, USA).

Statistical analysis and data visualization

To identify DEPs, the clusterProfiler R package (version 4.10.0) was employed. DEPs were selected based on the following criteria: a logFC greater than 1.5 or less than 0.67, along with a p-value of less than 0.05, as determined by the Student’s t-test or analysis of variance. A p-value threshold of 0.05 was considered indicative of statistical significance. For data visualization, the ComplexHeatmap package (version 2.18.0) was used to create a heatmap of DEPs, while the ggplot2 package (version 3.4.4) was utilized to generate the volcano plot.

Validation of biomarker candidates by ELISA

Biomarker candidates were quantified using Human ELISA kits from Jiangsu Meimian Industrial Co., Ltd. (Jiangsu, China), following the manufacturer’s protocol. ELISA plates were equilibrated at room temperature for 20 min, after which 50 µL of the standard solution was added to the designated standard wells, and 50 µL of the sample was added to the sample well, leaving the blank wells empty. Detection antibodies labeled with horseradish peroxidase (100 µL) were introduced into all wells, followed by incubation at 37 °C for 60 min. Following incubation, the liquid in the wells was discarded, and the wells were rinsed five times with 350 µL of washing solution. Substrates A and B (50 µL) were then added, and the plate was incubated in the dark at 37 °C for 15 min. The reaction was terminated with 50 µL of the stop solution, and the optical density at 450 nm was measured using a spectrophotometer within 15 min.