not-yet-known not-yet-known not-yet-known unknown 3.1. Differences in serum proteome profiles between OFC-positive and OFC-negative groups Patient characteristics are presented in Table 1. The median time to symptom onset in the OFC-positive group was 206.5 min. We found no difference in the characteristics of OFC-positive and OFC-negative patients. In both groups, IgE-dependent symptoms, such as urticaria or wheezing, did not develop during OFC. We collected serum from 9 of the 17 participants who provided consent to both serum and saliva collection . Of those, four were OFC-positive and five were OFC-negative. We identified 4,883 proteins via proteomic analysis of serum and analyzed 4,138 proteins after selecting proteins with at least two detected peptide counts. In subsequent group comparisons, the number of proteins for analysis was filtered to ~2,000 to ensure that at least one group contained a minimum of 70% valid values for each protein. First, we assessed differences in protein levels between the OFC-positive and OFC-negative groups before the OFC (Figure S1). We found significant differences in only 1.3% (23 proteins) of the 1,723 comparable proteins, suggesting that pre-OFC serum proteome profiles may not differ between OFC-positive and OFC-negative groups. Proteins with significantly altered expression 1 and 2 h after OFC and symptom onset compared with before OFC in each group are shown in Figure 1. At 1 h after OFC, 25 proteins (15 upregulated and 10 downregulated) in the OFC-positive group (Figure 1A) and 34 proteins (29 upregulated and five downregulated) in the OFC-negative group (Figure 1D) were detected. Contrastingly, 2 h after OFC, 614 proteins (609 upregulated and five downregulated) were observed in the OFC-positive group (Figure 1B) and 559 proteins (549 upregulated and 10 downregulated) were observed in the OFC-negative group (Figure 1E). The number of proteins with altered expression decreased to 309 (304 upregulated and 5 downregulated) at symptom onset in the OFC-negative group (Figure 1C). The upregulated proteins overlapped 2 h after OFC in the OFC-positive group (Serum-UpPOS2), 2 h after OFC in the OFC-negative group (Serum-UpNEG2), and at symptom onset in the OFC-positive group (Serum-UpS) (Figure S2). To further investigate the differences between Serum-UpPOS2 and Serum-UpNEG2, we performed a cluster analysis of enriched ontologies (Figure 2). Several terms and pathways, including neutrophil degranulation (R-HAS-6798695), were shared by the enriched ontologies in Serum-UpPOS2 and Serum-UpNEG2, suggesting that neutrophil degranulation may occur with or without symptom onset. The enriched ontologies in Serum-UpPOS2, but not in Serum-UpNEG2, included neddylation (R-HSA-8951664) and protein modification by small protein conjugation (GO: 0032446). In contrast, the enriched ontologies in Serum-UpNEG2, but not in Serum-UpPOS2, included focal adhesion (WP306) and platelet activation (hsa04611). The protein–protein interaction network and MCODEs of merged proteins of Serum-UpPOS2 and Serum-UpNEG2 (Figure 3) suggest that proteins contained in MCODE1 [PA700 (proteasome) complex, Table S1] and in MCODE4 (neddylation, Table S1) were upregulated predominantly in Serum-UpPOS2 but not in Serum-UpNEG2. Similarly, volcano plots of differentially expressed proteins relative to the time before OFC indicated transient upregulation of proteasome subunits 2 h after OFC in the OFC-positive group (Figure S3, S4). To verify these results, we measured serum proteasome 20S and NEDD8—protein modification molecules involved in neddylation—using ELISA and compared them before and 2 h after OFC (Figure S5). Changes in both serum proteasome 20S and NEDD8 levels were greater in the OFC-positive than in the OFC-negative group (p < 0.05). To focus on disease pathogenesis at FPIES symptom onset, we performed enrichment analysis (GO terms and KEGG pathways) using Enricher-KG on 73 proteins in Serum-UpS but not in Serum-UpPOS2 and Serum-UpNEG2 (indicated by white star in Figure S2). We identified genes related to neutrophil extracellular trap formation (KEGG: hsa04613), neutrophil activation pathways such as neutrophil degranulation (GO: 0043312), neutrophil activation involved in the immune response (GO: 0002283), and neutrophil-mediated immunity (GO: 0002446) (Figure 4).