Impact of inactivation methods on biosafety and antigen reactivity of Brucella melitensis from the perspective of Astral-DIA proteomics based on antibody immunoprecipitation mass spectrometry

Effective Brucella inactivation is essential for safe vaccine development, diagnostics, and sample handling, particularly in resource-limited regions without high-level biosafety facilities. This study evaluated heat (80°C/95°C, 10-20 min) and formaldehyde (0.4%/0.6%, 48-72 h) inactivation using the Rev.1 vaccine strain and three Brucella melitensis field isolates from Gansu (GS-XG1, GS-SN2, GS-MQ3). All field isolates were collected from a single geographic region (Gansu, China), which should be considered when interpreting the broader geographic generalizability of the findings. Complete inactivation of Rev.1 was achieved by heating at 80°C/95°C for 20 min or 0.6% formaldehyde for 48-72 h, with superior immunoreactivity compared to phenol inactivation as confirmed by ELISA and Western Blot. Field isolates showed greater resistance, surviving 80°C for 20 min and 0.4% formaldehyde for 72 h, requiring stricter conditions (95°C for 20 min or 0.6% formaldehyde for 72 h). Antibody immunoprecipitation-based Orbitrap Astral DIA proteomics covered ~60% of the proteome (~2,000/3,300 proteins) and identified 256, 311, and 318 differentially detected proteins (DDPs) for 80°C vs. 95°C heat, 48 h vs. 72 h formaldehyde, and heat vs. formaldehyde comparisons, respectively. These DDPs reflect inactivation-induced changes in protein detectability due to denaturation, aggregation, or cross-linking, rather than de novo gene expression (confirmed by metabolic inhibition assays showing complete absence of metabolic activity). Gene Ontology and KEGG analyses revealed that heat inactivation enriched cellular structure proteins while downregulating metabolic pathways, with 95°C potentially disrupting conformational epitopes. Formaldehyde treatment for 48 h better preserved soluble antigens and epitopes of ribosomal and regulatory proteins, whereas 72 h treatment caused greater cell envelope disruption. Protein-protein interaction networks indicated that heat inactivation enhanced immunoreactivity of membrane and stress proteins, making it suitable for targeted epitope studies, while formaldehyde preserved broader epitopes, benefiting whole-cell vaccines and multi-epitope screening. Inactivation protocols should be tailored to strain characteristics and intended applications. Astral-DIA proteomics provides molecular insights into antigenicity preservation, guiding future research on protein stability and epitope dynamics for improved brucellosis control.