Species-specific and processing-dependent fish allergen profile diversities: An integrated omics workflow for...

Seafood allergy is complex due to extensive species diversity, posing major challenges in food safety assessments, clinical diagnosis and dietary management. However, the absence of established workflows to resolve allergenomes limits correlations between allergen abundance, clinical sensitisation, and consumer risk. Mass spectrometry (MS)-based proteomics overcomes limitations of conventional immunoassay allergen detection by enabling unbiased protein identification and quantification, including allergen isoforms and low-abundance proteins within complex matrices. An integrated workflow combining immunological analyses, liquid chromatography-MS/MS proteomics, and bioinformatics was developed to characterise allergenomes across eight commonly consumed Asia-Pacific fish species. Comprehensive allergen profiles were established using in silico allergenicity predictions with AllerCatPro, combined with immunological validations using allergen-specific antibody and pooled patient sera. Across eight species, 529-1012 protein groups were identified, including all 11 fish muscle allergens, with pronounced interspecies differences in allergen composition and isoform distribution. The major fish pan-allergen parvalbumin was the most abundant allergen of most species and varied in abundance by up to 7-fold. Mackerel displayed a distinct low-parvalbumin profile with enriched metabolic allergens. Tissue heating induced a consistent shift toward enrichment of heat-stable and tissue-retained allergens, particularly parvalbumin, tropomyosin, and collagen. In contrast, heating of raw extracts generated more variable and species-specific retention of selected proteins, including heat-labile metabolic enzymes. In silico analysis predicted 14 proteins with strong allergenicity evidence for further validation. Fish allergenomes are species-specific and processing-dependent, positioning quantitative proteomics as a powerful platform for improved molecular risk assessment, and the development of representative diagnostic and food safety reference materials.