Specimen Inactivation Methods for Proteomics─Comparisons of Irradiation, Chemical, and Heat Treatments on Downstream Serum Analyses

Multiomic techniques, including proteomics, can provide novel insights for both pathogen detection and assessment of host responses to infection. Numerous studies have described the efficacy of heat inactivation, γ irradiation, or treatment with guanidinium-based chaotropic salts (e.g., TRIzol) for pathogen inactivation of biological samples to ensure biosafety and biosecurity. However, the effect of these methods on the greater serum proteome is less studied. Here, we sought to comprehensively measure the effects of four routinely used pathogen inactivation methods on the serum proteome of Rhesus macaques by characterizing the serum proteome pre-and-post inactivation treatment. Using the R-package MS-DAP for statistical benchmarking, our analysis revealed distinct changes in total peptide/protein detection and individual protein abundances (e.g., ALB, APOA1, and CRP) across inactivation methods and compared to their untreated controls. Specifically, we observed improved quantitative reproducibility in γ-irradiated samples across biological, technical, and experimental replicates compared to chemical inactivation and different heat combinations. These findings represent the first direct, experimental comparisons of effective pathogen inactivation methods on the serologic profiles of nonhuman primates and provide useful criteria for evaluating methods for biological specimen inactivation prior to proteomic analysis.