Shellfish allergy is a major global health concern, with prawns representing the most common trigger. Black tiger prawn (Penaeus monodon, BTP) and white leg prawn (Litopenaeus vannamei, WLP) dominate global consumption. Here, label-free shotgun proteomics combined with in-silico allergenicity prediction was applied to identify and compare the allergen profiles of BTP and WLP. Raw prawn extracts were analyzed by LC-MS/MS, and allergens were identified and predicted using AllerCatPro. The relative abundance of allergens was assessed using iBAQ%, and the differential abundance was evaluated using LFQ intensity. Although allergens accounted for only ∼4% of identified proteins, they represented 34-38% of total protein abundance, highlighting their immunological relevance. Myosin light chain was the most abundant allergen, followed by arginine kinase, sarcoplasmic calcium-binding protein, and tropomyosin in both species. Four low-abundance proteins were identified as novel allergen candidates. Notable differences in allergen profiles between the two species were observed at the level of allergen orthologs, isoforms, and variants, with nine uniquely identified in BTP and seven in WLP. Together, these findings reveal species-specific variations with implications for improved diagnostic strategies, therapeutic approaches, and allergen detection methods for shellfish allergy.

Allergic reactions to fish pose complex food safety challenges, driven by species diversity and underexplored intraspecies variability. This study comparatively examines allergen profiles in Malabar red snappers (Lutjanus malabaricus, n = 39) across body sizes, anatomical regions and production origins using SDS-PAGE, immunoblotting and quantitative mass spectrometry. Protein profiles varied greatly by fish size and muscle region, but not by origin. Smaller fish contained higher levels of major allergen parvalbumin and creatine kinase, while larger fish exhibited elevated levels of heat-labile allergens enolase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase. Parvalbumin levels were highest in head, followed by belly, dorsal, and tail. Greatest variation was observed for the three heat-stable allergens - parvalbumin, tropomyosin, and collagen. Minimal origin-dependent differences affected 2 of 11 registered fish allergens. We established an integrated proteomics workflow for systematic allergenicity assessments to uncover intraspecies variability and provide foundational knowledge for understanding intraspecies variability to improve food safety strategies.

Shellfish is a leading cause of life-threatening food anaphylaxis. Protein extraction is a critical step in allergenic protein (allergen) detection and quantification. This study evaluates the impact of protein extraction strategies on allergen detection in the two most widely consumed shellfish species, black tiger prawn (Penaeus monodon) and white leg prawn (Litopenaeus vannamei). Proteins were extracted from raw and boiled muscle tissues in four different buffers: urea-based, sodium dodecyl sulphate (SDS)-based, SDS-based with reducing agent, and phosphate-buffered saline (PBS), the most commonly used buffer in allergy investigations. Protein abundances were analysed using SDS-PAGE and label-free liquid chromatography-tandem mass spectrometry. Allergenicity was predicted using AllerCatPro and subsequently evaluated for antibody-based immunoreactivity. Protein extraction strategies significantly influenced proteome coverages, IgE-antibody binding, allergen diversities and relative abundances. Although PBS-based extracts yielded the highest overall relative allergen abundances (~84-95%), they generated the lowest allergen diversities and reduced relative abundances of key allergens, including tropomyosin and myosin light chain from raw tissues and arginine kinase from boiled tissues. In contrast, denaturing extracts enabled broader allergen representation, with urea-based extracts from boiled tissues generating the most comprehensive allergen repertoire. In addition to well-characterised allergens, up to 72 low-abundant proteins with strong bioinformatic evidence of allergenicity were identified, with their relative recovery varying across extraction strategies. Optimised protein extraction improves allergen detection workflows, supporting the discovery of underreported and low-abundant candidate allergens for future immunological validation, and facilitating more comprehensive risk assessment strategies.

Shellfish allergy is a major cause of food-induced severe adverse reactions worldwide, with shrimp representing one of the most implicated triggers. Detection and monitoring of allergenic proteins in shrimp-containing foods rely largely on immunoassay-based detection, typically targeting tropomyosin without species-level specificity. However, allergen composition may vary across species and processing conditions, limiting the accuracy of existing approaches. Here, we applied quantitative proteomics to characterise allergen profiles in five commonly consumed shrimp species under raw and heat-treated conditions. Protein extracts (n = 3 per species/treatment) were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following in-solution digestion. Identified proteins were mapped against curated allergen databases and species-specific transcriptomes to assess presence and relative abundance. Complementary SDS-PAGE and immunoblot analyses using allergen-specific antibodies were performed to evaluate qualitative detection across species and treatments. Proteomic analysis revealed marked interspecies variability in allergen presence and abundance. Several allergens exhibited differential stability following heat treatment, reflecting distinct biophysical properties influencing persistence in heat-processed foods. Selected putative allergens identified by transcriptome analysis, including aldolase and enolase, were confirmed at the protein level, while others were not detected. Moreover, weak concordance between transcriptomic and proteomic abundance was observed for multiple allergens, including arginine kinase and myosin light chain. Immunoblotting for tropomyosin and myosin light chain demonstrated inconsistent detection across species and treatments, highlighting limitations of antibody-based detection systems. These findings establish that shrimp allergen composition is species- and heat processing-dependent, with implications for risk assessment frameworks. Quantitative proteomics provides a robust platform for comprehensive allergen profiling and supports improved detection strategies for aquatic food safety.

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.

Food fraud poses a serious safety risk and affects the economy, with seafood being particularly vulnerable due to high species diversity and complex global supply chains. Accurate fish species identification is crucial for sustainable fishery management, food safety and protecting consumers with species-specific fish allergies who are at risk of life-threatening anaphylaxis. While some methods such as DNA-based PCR are well-established, they have limitations for processed foods and are costly, complex and time-consuming. Glycan markers are highly stable and have recently emerged as a tool for food authentication due to their unique species-specific characteristics. This study introduces N-glycan profiling as a novel technique for fish species authentication and addresses the need for reliable methods applicable to processed seafood products. By employing liquid chromatography ion mobility-mass spectrometry analysis, we examined N-glycan profiles of raw and heated fish muscle tissues from three fish species, which represent widely consumed seabass and snapper as well as their potential counterfeit substitute, tilapia from markets and restaurants. N-glycan structures containing different degrees of O-acetylated sialic acids (O-Ac-Sias) were identified as species-specific markers and clustering based on their percentage abundance enabled species classification. This study provides the foundation for the development of a rapid, species-specific authentication tool, which could be employed throughout the seafood supply chain, from harvest to retail, improving traceability and reducing mislabeling in markets and restaurants.

Shellfish allergy, triggered by immune reactions to crustacean and mollusk proteins upon consumption/inhalation, is one of the most severe and persistent food allergies, affecting approximately 1

IntroductionFish is a major food allergy trigger with a complex variety of allergenic protein isoforms and vast species diversity exhibiting variable allergenicity. This is the first study to systematically compile fish isoallergen and variant entries associated with ingestion-related allergic reactions.MethodsEntries were compiled from four major allergen databases: World Health Organization and International Union of Immunological Societies (WHO/IUIS), AllergenOnline, Comprehensive Protein Allergen Resource (COMPARE), and Allergome, including evidence from in vitro IgE-binding assays and complete amino acid sequences. Challenges in predicting the allergenicity of fish isoallergens and variants were evaluated, and the sensitivity of five widely used in silico tools (AllerCatPro 2.0, AlgPred 2.0, pLM4Alg, AllergenFP v.1.0, and AllerTop v.2.0) was assessed. Epitope mapping and phylogenetic analyses were performed for the major fish allergen parvalbumin, incorporating experimentally validated B-cell epitope data from the Immune Epitope Database (IEDB) and evolutionary relationships.ResultsA comprehensive dataset of 79 unique fish isoallergen and variant entries from 34 fish species was identified, with 25 entries common across all four databases. AllerCatPro 2.0 achieved the highest sensitivity (97.5%). A phylogenetic tree was constructed, integrating epitope data to optimize protein family-specific thresholds for differentiating allergenic from less/non-allergenic parvalbumins. A threshold of ≥4 IEDB-mapped epitopes allowing up to two mismatches captured 52 out of 54 parvalbumin sequences (96%) in the dataset, effectively distinguishing between parvalbumin classes.DiscussionThis study enhances understanding of fish allergy by systematically compiling fish isoallergens and variants and integrating B-cell epitope data. The optimized thresholds improve the performance of allergenicity prediction tools and can be applied to other protein families in future studies.

Mung bean is an increasingly cultivated legume. This study compared mung bean varieties 'KPS2' from Thailand (Th) and 'Imara' from Tanzania (T) with a focus on protein composition, allergenicity, and techno-functional properties. Two rounds alkaline-acid extraction were performed to produce mung bean protein isolate (MBPI Th1/T1 and Th2/T2), supernatant (S) and protein-poor residue (PPR). Mass spectrometric analysis revealed high abundance of 8 s-vicilin and 11 s-legumin in MBPI and S. Extraction removed considerable amounts of the seed albumin allergen but increased the relative abundance of cupins in MBPI. Higher vicilin levels were found in Th1 samples, contributed to increased protein solubility above pH 6.5. Th formed stronger gels which were more stable at higher frequencies. In contrast, T proteins were structurally more flexible, leading to its improved foaming ability. This study provides the knowledge and methods for appropriate selection of mung bean varieties for various food applications.

Spotted seabass (Lateolabrax maculatus) is the second largest maricultural fish species in China and is the main trigger of food-related allergic reactions. Nevertheless, studies on the allergens of L. maculatus are limited. This study aimed to characterize pan-allergen parvalbumin from L. maculatus. Two proteins of about 11 kDa were purified and confirmed as parvalbumins by mass spectrometry. The IgG- and IgE-binding activities were evaluated through an immunoblotting assay. The molecular characteristics of β-parvalbumin were investigated by combining proteomics, genomics, and immunoinformatics approaches. The results indicated that β-parvalbumin consists of 109 amino acids with a molecular weight of 11.5 kDa and is the major allergen displaying strong IgE-binding capacity. In silico analysis and a dot blotting assay confirmed seven linear B cell epitopes distributed mainly on α-helixes and the calcium-binding loops. In addition, the cross-reactivity among 26 commonly consumed fish species was analyzed. The in-house generated anti-L. maculatus parvalbumin polyclonal antibody recognized 100% of the 26 fish species, demonstrating cross-reactivity and better binding capacity than the anticod parvalbumin antibody. Together, this study provides an efficient protocol to characterize allergens with multiomics methods and supports parvalbumin from L. maculatus as a candidate for fish allergen determination and allergy diagnosis.

Small calcium-binding proteins such as parvalbumins (PVs) are major seafood and fish allergens. However, the impact of structural changes on their capacity to bind IgE has not been studied in detail. Therefore, fish and reptilian PVs, as well as human α-PV, were selected for biochemical, structural, and IgE binding studies. Likely due to their high solubility, crystallization proved difficult, so additional techniques were used to promote crystallization of the proteins. Novel crystal structures were determined for human PV, cod allergen Gad m 1.0201, saltwater crocodile allergen Cro p 1.0101, and the α-PV from thornback ray. β-PVs are considered the major fish allergens, while α-PVs are rarely categorized as allergens. To explain these differences, the results of structural and IgE binding studies were combined. This approach allowed us to provide new insight into IgE binding epitopes present on PVs, focusing on cross-reactivity among the selected α- and β-PVs. In addition, we have shown that these proteins display remarkable thermal stability across a range of pH conditions, which is relevant in the case of food allergens and food processing. Moreover, it is shown that the presence of calcium cations is critical for stability of the studied PVs via their protein folding, which has an impact on the formation of IgE binding epitopes. These studies shows the stability of fish and reptile PV allergens, and it allows for further evaluation of their IgE cross-reactivity.

Due to the widespread use of shellfish ingredients in food products, accurate food labelling is urgently needed for consumers with shellfish allergies. Most crustacean allergen detection systems target the immunorecognition of the allergenic protein tropomyosin. However, this mode of detection may be affected by an origin-dependent protein composition. This study determined if the geographic location of capture, or aquaculture, influenced the allergenic protein profiles of Black Tiger Shrimp (Penaeus monodon), one of the most farmed and consumed shrimp species worldwide. Protein composition was analysed in shrimp from nine different locations in the Asia–Pacific by SDS-PAGE, immunoblotting, and mass spectrometry. Ten of the twelve known shrimp allergens were detected, but with considerable differences between locations. Sarcoplasmic calcium-binding protein, myosin light chain, and tropomyosin were the most abundant allergens in all locations. Hemocyanin-specific antibodies could identify up to six different isoforms, depending on the location of origin. Similarly, tropomyosin abundance varied by up to 13 times between locations. These findings suggest that allergen abundance may be related to shrimp origin and, thus, shrimp origin might directly impact the readout of commercial crustacean allergen detection kits, most of which target tropomyosin, and this should be considered in food safety assessments.

SCOPE:Edible insect proteins are increasingly introduced as an alternative sustainable food source to address the world's need to feed the growing population. Tropomyosin is the main insect allergen; however, additional potential allergens are not well characterized and the impact of extraction procedures on immunological reactivity is unknown. METHODS AND RESULTS:Proteins from different commercial food products derived from cricket (Acheta domesticus) and black soldier fly (BSF) (Hermetia illucens) are extracted using five different extraction buffers. The proteins are analyzed by SDS-PAGE and immunoblotting using allergen-specific antibodies and crustacean allergic patient sera. IgE binding bands are analyzed by mass spectrometry as well as the complete allergen profile of all 30 extracts. Urea-based buffers are most efficient in extracting insect allergens. Shrimp-specific antibody cross-reactivity to tropomyosin from cricket and BSF indicates high sequence and structural similarity between shrimp and insects. Additional unique allergens are identified in both species, including hemocyanin, vitellogenin, HSP20, apolipophorin-III, and chitin-binding protein. CONCLUSIONS:Identifying potential allergenic proteins and their isoforms in cricket and BSF requires specific extraction approaches using urea-based methods. While tropomyosin is the most abundant and immunoreactive allergen, seven unique allergens are identified, highlighting the need for insect species-specific allergen detection in food products.

Integrated bioinformatics tools have created more efficient and robust methods to overcome in vitro challenges and have been widely utilized for the investigation of food proteins and the generation of peptide sequences. This study aimed to analyze the physicochemical properties and bioactivities of novel peptides derived from hydrolyzed milkfish (Chanos chanos) protein sequences and to discover their potential angiotensin-converting enzyme (ACE)- and dipeptidyl peptidase-4 (DPPIV)-inhibitory activities using machine learning-based tools, including BIOPEP-UWM, PeptideRanker, and the molecular docking software HADDOCK 2.4. Nine and three peptides were predicted to have ACE- and DPPIV-inhibitory activities, respectively. The DPPIV-inhibitory peptides were predicted to inhibit the compound with no known specific mode. Meanwhile, two tetrapeptides (MVWH and PPPS) were predicted to possess a competitive mode of ACE inhibition by directly binding to the tetra-coordinated Zn ion. Among all nine discovered ACE-inhibitory peptides, only the PPPS peptide satisfied the drug-likeness analysis requirements with no violations of the Lipinski rule of five and should be further investigated in vitro.