Food analysis can be challenging due to the nature of the samples: often multiple species, high lipid or carbohydrate content, and typically solid matrices are just some of the obstacles. For protein analysis, the S-Trap sample preparation system has revolutionized proteomics by delivering robust, repeatable protein extraction and digestion, even with such difficult, complex mixtures. S-Trap analysis has been applied to a wide range of substrates, including foods, to study allergens.
LCMS detection of allergens has two major advantages over ELISA techniques: denaturation of epitopes, for example through the cooking process, does not interfere with detection, and assays can be multiplexed with extreme ease. S-Trap sample preparation is a robust, easy-to-execute, and rapid technique suitable for reliably detecting trace amounts of allergens in many kinds of foods, substances, and matrices. The integrated approach thus enables the widespread use of LCMS in assessing allergenic hazards. Reliable S-Trap sample processing helps ensure food safety and address the needs of individuals with severe allergies. Several select studies follow.
The S-Trap system enabled the identification and semi-quantification of 4,586 proteins from 12 different allergenic source materials including foods. Multiple known allergens were detected, such as Ara h 1 and Ara h 3 in peanuts, Cor a 9 and Cor a 14 in hazelnuts, Fel d 1 and Can f 1 in pet dander, and Der p proteins from dust mites, among others. In total, 129 specific allergens were identified from the 12 sources. The total relative abundance of source-specific allergens ranged between 0.8% (lamb’s quarters) and 63% (olive pollen).
The system was sensitive enough that allergens from other species were identified while analyzing house dust mites, including allergens from sheep scab mites (Pso o 10), mold mites (Tyr p 13 lipocalin and Tyr p 28 actin-like), blood fluke parasites (cysteine protease allergens Sch ma and Sm31), and cockroaches (NAD(P)-binding allergen Per a 13). These exogenous species comprised more than 18% of the studied material.
On average, 16% of proteins identified were predicted to be strongly allergenic. This includes those from peanut (594 total proteins identified, of which 89 have strong evidence of being allergenic), lamb's quarters pollen (560, 57 allergenic), hazelnut (554, 71), Johnson grass pollen (540, 87), mugwort (441, 70), birch (440, 54), ragweed (436, 63), dog (310, 59), cat (286, 42), dust mite (266, 73), Timothy grass pollen (117, 39), and olive (42, 20). Additionally, 20% of identified proteins from all sources were predicted to be weakly allergenic.
Cashew nuts can elicit mild to severe allergic reactions, and three known allergenic cashew proteins exist. Similar reactions are caused by pathogenesis‐related protein 10 (PR10) allergens from pollen, and this study sought and identified previously unknown PR10-like proteins in cashew nuts. Samples were processed by S-Traps, and LCMS identified three novel PR10-like proteins. Immunoblotting and RNAseq verified all three, and in silico analysis predicted them all to be allergenic. Cashew nut PR10-like proteins show significant homology to known nut, seed, and other allergens like Pru av 1 from cherry. These three PR10-like cashew proteins thus represent new potential isoallergens in cashews.
In the study of allergens, LCMS has advantages over ELISA especially for processed foods, as the recognized epitopes of allergens are frequently altered or destroyed by heat. This study sought to develop and compare LC-MS/MS and real-time PCR methods for detecting silkworm (Bombyx mori) analyzed 24 commercially available food products and 19 species including edible insects, crustaceans, and mollusks. S-Trap processing allowed the identification of 2016 peptides (5435 peptide spectrum matches, PSMs) and 760 distinct proteins including known allergenic proteins such as arginine kinase, tropomyosin, alpha-amylase, peroxiredoxin, hemolymph lipoprotein, and other lipoproteins. Thirteen silkworm-specific peptides were found that have no cross-reactivity with other species.
Using cookies as a food model, proteomics could detect silkworm at concentrations as low as 0.0005%--a value close to 10 parts per billion (ppb)--with high linearity (R² = 0.995) and an average CV of 5%. This outperforms real-time PCR with a detection limit of 0.001%, and contrasts to detection limits around 1% - 0.1% in previous studies.
Other species analyzed included silkworm (B. mori), mealworm (Tenebrio molitor), rhinoceros beetle larvae (Trypoxylus dichotomus), white flower chafer beetle larvae (Protaetia brevitarsis), rice grasshopper (Oxya chinensis), two-spotted cricket (Gryllus bimaculatus), supermealworm (Zophobas atratus), migratory locust (Locusta migratoria), honeybee pupae (Apis mellifera ligustica), oriental garden cricket (Teleogryllus emma), house cricket (Acheta domesticus), manila clam (Venerupis philippinarum), ocean quahog (Arctica islandica), abalone (Haliotidae haliotis), mussel (Mytilus coruscus), oyster (Crassostrea gigas), whiteleg shrimp (Litopenaeus vannamei), crab (Portunus trituberculatus), and crayfish (Homarus americanus).
The S-Trap system facilitated the identification and quantification of allergenic proteins in Black Tiger Shrimp (Penaeus monodon) originating from different geographical locations. The study identified ten of the twelve known shrimp allergens, whose abundance varied significantly between different shrim origins. Major identified allergens include tropomyosin, with abundance variations up to 13-fold between locations, myosin light chain (MLC) 1 and 2, and sarcoplasmic calcium-binding protein (SCBP). Together these proteins contribute more than 50% of the observed protein abundance. Isoallergens of tropomyosin and SCBP were also found among the 253 to 648 proteins identified from shrimp coming from different locations. The location-dependence of both allergen content and isoallergen profiles suggests environmental, and potentially genetic, influences. Importantly, because most commercial detection kits target tropomyosin, the origin of the shrimp may directly impact the accuracy of crustacean allergen detection.
The S-Trap system was utilized to prepare samples of cat dander extract (CDE) to obtain a comprehensive allergenic protein profile. In total, 4,492 CDE peptides were identified, and their removal or degradation by different air purification technologies was assessed. The study found that 2,731 peptides, or 61% of the CDE peptides, were completely degraded by photoelectrochemical oxidation (PECO) upon exposure to UV-A for one hour; this far exceeded HEPA filtration alone, which showed an 8% reduction.
The S-Trap system enabled the identification of over 7,000 different peptides corresponding to 1,385–2,664 proteins in mung bean extracts. The mung bean allergen 8S globulin (termed Vig r 2 for mung beans) was the most abundant, and other major allergens such as 8S-vicilin (Vig r 4) and pathogenesis-related protein (Vig r 1) were also detected. Notably, the protein composition varied between samples from Tanzania and Thailand, offering insights into how cultivation locations can influence the properties of mung beans. This study highlights the S-Trap system's capability to provide detailed protein profiles, contributing to the understanding of allergenicity in different food sources.
In conclusion, the S-Trap system enables the examination of allergens in food samples by reproducibly producing LC-MS-ready samples from all kinds of difficult matrices. This capability ensures high sensitivity and comprehensive analysis, making the S-Trap an invaluable tool for allergen detection.