Biomarker Discovery in Minipigs Reveals Sex- and Survival-Associated Proteomic Signatures

Large-scale radiological or nuclear incidents pose a significant public health threat, requiring reliable biomarkers to quickly assess radiation exposure. Proteins are promising candidates due to their dynamic response to ionizing radiation and their detectability in clinical settings. We studied protein profiles linked to minipig survival and investigated sex-dependent differences in protein expression after total-body irradiation (TBI). In this label-free quantitative proteomics study, peripheral blood samples were collected from 27 Göttingen minipigs across three sequential experimental series to identify potential radiation-responsive protein biomarkers. Samples were obtained from each minipig at four time points: 3 days before irradiation (−3 days, A group) and 1, 3, and 10 days after 2.1 Gy total-body irradiation (B, C and D groups, respectively). A label-free relative quantification method was employed, utilizing an RP-nanoLC-ESI-MS/MS system with a Q Exactive mass spectrometer (Thermo). First, plasma was separated from the blood samples. Proteins were then depleted, reduced, alkylated, and enzymatically digested with trypsin/LysC following standard procedures. Protein identification was performed using MaxQuant, Perseus, and RStudio. Statistical significance was determined using a t-test, with P ≤ 0.05 considered significant. Data are available via ProteomeXchange with identifier PXD071774 (DOI: 10.6019/PXD071774). Comparative proteomic profiling revealed differential regulation of many proteins related to radiation exposure, survival, and sex-specific effects. Based on these findings, the top 18 candidates strongly linked to gamma radiation were selected. These proteins are involved in hemostasis, inflammatory signaling, immune regulation, and tissue repair. Validation across three independent studies confirmed the reproducibility of key biomarkers. Plasma proteomics also showed distinct sex-specific expression patterns after irradiation. In males, hemoglobin subunits alpha and beta (HBA/HBB) were significantly upregulated (log 2 fold change >2), while many Ig-like domain proteins and immunoglobulin heavy-chain components were consistently downregulated. In females, fibrinogen alpha and beta chains (FGA/FGB), complement C3, and apolipoprotein E (APOE) showed notable increases. Both sexes exhibited decreases in lysozyme C-3, serotransferrin, and von Willebrand factor, with a larger reduction observed in females. When stratifying proteomics data by survival, a clear plasma signature associated with outcomes was identified. Non-survivors had higher periostin levels (the most prominent signal) and elevated lipopolysaccharide-binding protein, while survivors showed higher levels of immune proteins such as Ig lambda chain and complement C1q subunits. These differences appeared shortly after irradiation, persisted over time, and remained consistent across different experimental batches. Our findings support the usefulness of minipigs as a translational large-animal model for radiation biomarker discovery. The identified plasma proteins are promising candidates for further validation as indicators of radiation injury and potential predictors of clinical outcomes.