How α-Helical Motifs Form Functionally Diverse Lipid-Binding Compartments.

Lipids are produced site-specifically in cells and then distributed nonrandomly among membranes via vesicular and nonvesicular trafficking mechanisms. The latter involves soluble amphitropic proteins extracting specific lipids from source membranes to function as molecular solubilizers that envelope their insoluble cargo before transporting it to destination sites. Lipid-binding and lipid transfer structural motifs range from multi-ß-strand barrels, to ß-sheet cups and baskets covered by α-helical lids, to multi-α-helical bundles and layers. Here, we focus on how α-helical proteins use amphipathic helical layering and bundling to form modular lipid-binding compartments and discuss the functional consequences. Preformed compartments generally rely on intramolecular disulfide bridging to maintain conformation (e.g., albumins, nonspecific lipid transfer proteins, saposins, nematode polyprotein allergens/antigens). Insights into nonpreformed hydrophobic compartments that expand and adapt to accommodate a lipid occupant are few and provided mostly by the three-layer, α-helical ligand-binding domain of nuclear receptors. The simple but elegant and nearly ubiquitous two-layer, α-helical glycolipid transfer protein (GLTP)-fold now further advances understanding.

From allergen genes to allergy vaccines.

IgE-mediated allergy is a hypersensitivity disease affecting more than 25% of the population. The structures of the most common allergens have been revealed through molecular cloning technology in the past two decades. On the basis of this knowledge of the sequences and three-dimensional structures of culprit allergens, investigators can now analyze the immune recognition of allergens and the mechanisms of allergic inflammation in allergic patients. Allergy vaccines have been constructed that are able to selectively target the aberrant immune responses in allergic patients via different pathways of the immune system. Here we review various types of allergy vaccines that have been developed based on allergen structures, results from their clinical application in allergic patients, and future strategies for allergen-specific immunotherapy and allergy prophylaxis.

Pioneering bioinformatics with agent-based modelling: an innovative protocol to accurately forecast skin or respiratory allergic reactions to chemical sensitizers.

The assessment of the allergenic potential of chemicals, crucial for ensuring public health safety, faces challenges in accuracy and raises ethical concerns due to reliance on animal testing. This paper presents a novel bioinformatic protocol designed to address the critical challenge of predicting immune responses to chemical sensitizers without the use of animal testing. The core innovation lies in the integration of advanced bioinformatics tools, including the Universal Immune System Simulator (UISS), which models detailed immune system dynamics. By leveraging data from structural predictions and docking simulations, our approach provides a more accurate and ethical method for chemical safety evaluations, especially in distinguishing between skin and respiratory sensitizers. Our approach integrates a comprehensive eight-step process, beginning with the meticulous collection of chemical and protein data from databases like PubChem and the Protein Data Bank. Following data acquisition, structural predictions are performed using cutting-edge tools such as AlphaFold to model proteins whose structures have not been previously elucidated. This structural information is then utilized in subsequent docking simulations, leveraging both ligand-protein and protein-protein interactions to predict how chemical compounds may trigger immune responses. The core novelty of our method lies in the application of UISS-an advanced agent-based modelling system that simulates detailed immune system dynamics. By inputting the results from earlier stages, including docking scores and potential epitope identifications, UISS meticulously forecasts the type and severity of immune responses, distinguishing between Th1-mediated skin and Th2-mediated respiratory allergic reactions. This ability to predict distinct immune pathways is a crucial advance over current methods, which often cannot differentiate between the sensitization mechanisms. To validate the accuracy and robustness of our approach, we applied the protocol to well-known sensitizers: 2,4-dinitrochlorobenzene for skin allergies and trimellitic anhydride for respiratory allergies. The results clearly demonstrate the protocol's ability to differentiate between these distinct immune responses, underscoring its potential for replacing traditional animal-based testing methods. The results not only support the potential of our method to replace animal testing in chemical safety assessments but also highlight its role in enhancing the understanding of chemical-induced immune reactions. Through this innovative integration of computational biology and immunological modelling, our protocol offers a transformative approach to toxicological evaluations, increasing the reliability of safety assessments.

Efficient experimental method for purifying allergens from aqueous extracts.

Studying the molecular and immunological basis of allergic diseases often requires purified native allergens. The methodologies for protein purification are usually difficult and may not be completely successful. The objective of this work was to describe a methodology to purify allergens from their natural source, while maintaining their native form. The purification strategy consists of a three-step protocol and was used for purifying five specific allergens, Ole e 1, Amb a 1, Alt a 1, Bet v 1 and Cup a 1. Total proteins were extracted in PBS (pH 7.2). Then, the target allergens were pre-purified and enriched by salting-out using increasing concentrations of ammonium sulfate. The allergens were further purified by anion exchange chromatography. Purification of Amb a 1 required an extra step of cation exchange chromatography. The detection of the allergens in the fractions obtained were screened by SDS-PAGE, and Western blot when needed. Further characterization of purified Amb a 1 was performed by mass spectrometry. Ole e 1, Alt a 1, Bet v 1 and Cup a 1 were obtained at > 90 % purity. Amb a 1 was obtained at > 85 % purity. Overall, we propose an easy-to-perform purification approach that allows obtaining highly pure allergens. Since it does not involve neither chaotropic nor organic reagents, we anticipate that the structural and biological functions of the purified molecule remain intact. This method provides a basis for native allergen purification that can be tailored according to specific needs.

Structural analysis of human IgE monoclonal antibody epitopes on dust mite allergen Der p 2.

BACKGROUND: Human IgE (hIgE) mAbs against major mite allergen Der p 2 developed using human hybridoma technology were used for IgE epitope mapping and analysis of epitopes associated with the hIgE repertoire. OBJECTIVE: We sought to elucidate the new hIgE mAb 4C8 epitope on Der p 2 and compare it to the hIgE mAb 2F10 epitope in the context of the allergenic structure of Der p 2. METHODS: X-ray crystallography was used to determine the epitope of anti-Der p 2 hIgE mAb 4C8. Epitope mutants created by targeted mutagenesis were analyzed by immunoassays and in vivo using a human high-affinity IgE receptor (FcεRIα)-transgenic mouse model of passive systemic anaphylaxis. RESULTS: The structure of recombinant Der p 2 with hIgE mAb 4C8 Fab was determined at 3.05 Å. The newly identified epitope region does not overlap with the hIgE mAb 2F10 epitope or the region recognized by 3 overlapping hIgE mAbs (1B8, 5D10, and 2G1). Compared with wild-type Der p 2, single or double 4C8 and 2F10 epitope mutants bound less IgE antibodies from allergic patients by as much as 93%. Human FcεRIα-transgenic mice sensitized by hIgE mAbs, which were susceptible to anaphylaxis when challenged with wild-type Der p 2, could no longer cross-link FcεRI to induce anaphylaxis when challenged with the epitope mutants. CONCLUSIONS: These data establish the structural basis of allergenicity of 2 hIgE mAb nonoverlapping epitopes on Der p 2, which appear to make important contributions to the hIgE repertoire against Der p 2 and provide molecular targets for future design of allergy therapeutics.

Novel machine learning method allerStat identifies statistically significant allergen-specific patterns in protein sequences.

Cutting-edge technologies such as genome editing and synthetic biology allow us to produce novel foods and functional proteins. However, their toxicity and allergenicity must be accurately evaluated. It is known that specific amino acid sequences in proteins make some proteins allergic, but many of these sequences remain uncharacterized. In this study, we introduce a data-driven approach and a machine-learning method to find undiscovered allergen-specific patterns (ASPs) among amino acid sequences. The proposed method enables an exhaustive search for amino acid subsequences whose frequencies are statistically significantly higher in allergenic proteins. As a proof-of-concept, we created a database containing 21,154 proteins of which the presence or absence of allergic reactions are already known and applied the proposed method to the database. The detected ASPs in this proof-of-concept study were consistent with known biological findings, and the allergenicity prediction performance using the detected ASPs was higher than extant approaches, indicating this method may be useful in evaluating the utility of synthetic foods and proteins.

Structural homology of mite profilins to plant profilins is not indicative of allergic cross-reactivity.

Structural and allergenic characterization of mite profilins has not been previously pursued to a similar extent as plant profilins. Here, we describe structures of profilins originating from Tyrophagus putrescentiae (registered allergen Tyr p 36.0101) and Dermatophagoides pteronyssinus (here termed Der p profilin), which are the first structures of profilins from Arachnida. Additionally, the thermal stabilities of mite and plant profilins are compared, suggesting that the high number of cysteine residues in mite profilins may play a role in their increased stability. We also examine the cross-reactivity of plant and mite profilins as well as investigate the relevance of these profilins in mite inhalant allergy. Despite their high structural similarity to other profilins, mite profilins have low sequence identity with plant and human profilins. Subsequently, these mite profilins most likely do not display cross-reactivity with plant profilins. At the same time the profilins have highly conserved poly(l-proline) and actin binding sites.

Electrophilic Reactivity of Sulfated Alcohols in the Context of Skin Sensitization.

The surfactant sodium lauryl sulfate (SLS), although consistently positive in the murine local lymph node assay (LLNA) for skin sensitization, shows no evidence of being a human sensitizer and is often described as a false positive, lacking structural alerts for sensitization. However, there is evidence of the cinnamyl sulfate anion being the metabolite responsible for the sensitization potential of cinnamyl alcohol to humans and in animal tests. Here, manufacturing chemistry data and physical organic chemistry principles are applied to confirm that SLS is not reactive enough to sensitize, whereas sensitization to cinnamyl alcohol via cinnamyl sulfate is plausible. Sensitization data for several other primary alcohols, including geraniol, farnesol, and possibly hydrocortisone, are also consistent with this mechanism. It seems possible that biosulfation may play a wider role than has previously been recognized in skin sensitization.

Can Physicochemical Properties Alter the Potency of Aeroallergens? Part 2 - Impact of Physicochemical Properties.

PURPOSE OF REVIEW: A holistic perspective on how physicochemical properties modulate the allergenicity of proteins has recently been performed for food allergens, launching the challenge of a similar analysis for aeroallergens. After a first review on aeroallergen classification into protein families (Part 1), this second part (Part 2) will exploit the impact of physicochemical properties (abundance/biological function, protein structure/presence of post-translational modifications, ligand/cofactor/lipid-binding) on inhalant protein allergenicity. RECENT FINDINGS: The abundance linked to biological function is correlated with increased allergenic risk for most protein families, while the loss of structural integrity with consequent destruction of conformational epitopes is well linked with decreased allergenicity. Ligand-binding effect totally depends on the ligand type being highly variable among aeroallergens. Knowledge about the physicochemical properties of aeroallergens is still scarce, which highlights the need for research using integrated approaches (in silico and experimental) to generate and analyze new data on known/new aeroallergens.

AllerCatPro 2.0: a web server for predicting protein allergenicity potential.

Proteins in food and personal care products can pose a risk for an immediate immunoglobulin E (IgE)-mediated allergic response. Bioinformatic tools can assist to predict and investigate the allergenic potential of proteins. Here we present AllerCatPro 2.0, a web server that can be used to predict protein allergenicity potential with better accuracy than other computational methods and new features that help assessors making informed decisions. AllerCatPro 2.0 predicts the similarity between input proteins using both their amino acid sequences and predicted 3D structures towards the most comprehensive datasets of reliable proteins associated with allergenicity. These datasets currently include 4979 protein allergens, 162 low allergenic proteins, and 165 autoimmune allergens with manual expert curation from the databases of WHO/International Union of Immunological Societies (IUIS), Comprehensive Protein Allergen Resource (COMPARE), Food Allergy Research and Resource Program (FARRP), UniProtKB and Allergome. Various examples of profilins, autoimmune allergens, low allergenic proteins, very large proteins, and nucleotide input sequences showcase the utility of AllerCatPro 2.0 for predicting protein allergenicity potential. The AllerCatPro 2.0 web server is freely accessible at https://allercatpro.bii.a-star.edu.sg.

The dimer effect: A refinement approach towards skin sensitization assessment in-chemico using Amino acid Derivative Reactivity Assay.

Skin sensitization is a key endpoint for safety assessment, especially for cosmetics and personal care products. The adverse outcome pathway for skin sensitization and the chemical and biological events driving the induction of human skin sensitization are now well understood. Several non-animal test methods have been developed to predict sensitizer potential by measuring the impact of chemical sensitizers on these key events. In this work, we have focused on Key Event 1 (the molecular initiating step), which is based on formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. There exists three in-chemico assays approved by the Organization for Economic Co-operation and Development-(1) Direct Peptide Reactivity Assay (DPRA), (2) Amino Acid Derivative Reactivity Assay (ADRA), and (3) Kinetic Direct Peptide Reactivity Assay (kDPRA) to quantify peptide/amino acid derivative depletion after incubation with test chemicals. However, overestimated depletion of the cysteine-based peptide/amino acid derivatives is known in such assays because of the dimerization of the thiol group. In this present work, we report the synthesis and structural confirmation of the dimer of N-(2-[1-naphthyl]acetyl)-L-cysteine (NAC) from the ADRA assay to allow simultaneous determination of (a) peptide depletion by quantifying NAC monomer and (b) peptide dimerization by quantifying NAC dimer thereby eliminating the overestimation. We present a case study with three chemicals to demonstrate the importance of this approach. Thus, this simultaneous assay gives a more informed view of the peptide reactivity of chemicals to better identify skin sensitizers.

IgE cross-inhibition between Ara h 1 and Ara h 2 is explained by complex formation of both major peanut allergens.

BACKGROUND: Surprisingly, IgE cross-reactivity between the major peanut allergens Ara h 1, 2, and 3 has been reported despite very low sequence identities. OBJECTIVE: We investigated the unexpected cross-reactivity between peanut major allergens. METHODS: Cross-contamination of purified natural Ara h 1, 2, 3, and 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot test, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and sandwich enzyme-linked immunosorbent assay (ELISA). IgE cross-reactivity was studied with sera of peanut-allergic patients (n = 43) by ELISA and ImmunoCAP inhibition using both intact natural and recombinant allergens and synthetic peptides representing postulated Ara h 1 and Ara h 2 cross-reactive epitopes. RESULTS: Both purified nAra h 1 and nAra h 3 were demonstrated to contain small but significant amounts of Ara h 2 and Ara h 6 (<1%) by sandwich ELISA, SDS-PAGE/Western blot analysis, and LC-MS/MS. IgE cross-inhibition between both 2S albumins and Ara h 1 and Ara h 3 was only observed when using natural purified allergens, not recombinant allergens or synthetic peptides. Apparent cross-reactivity was lost when purified nAra h 1 was pretreated under reducing conditions, suggesting that Ara h 2 and Ara h 6 contaminations may be covalently bound to Ara h 1 via disulfide interactions. CONCLUSION: True cross-reactivity of both peanut 2S albumins with Ara h 1 and Ara h 3 could not be demonstrated. Instead, cross-contamination with small quantities was shown to be sufficient to cause significant cross-inhibition that can be misinterpreted as molecular cross-reactivity. Diagnostic tests using purified nAra h 1 and nAra h 3 can overestimate their importance as major allergens as a result of the presence of contaminating 2S albumins, making recombinant Ara h 1 and Ara h 3 a preferred alternative.

Purified Clinoptilolite-Tuff as an Efficient Sorbent for Food-Derived Peanut Allergens.

The avoidance of allergen intake is crucial for persons affected by peanut allergy; however, the cross-contamination of food is common and leads to unpredictable consequences after the consumption of supposedly "safe" food. The aim of the present study was to eliminate harmful traces of peanut allergens from food using purified clinoptilolite-tuff (PCT)-a specially processed zeolite material. Analyses were performed using a peanut ELISA and a Coomassie blue (Bradford) assay. Mimicking conditions of the human gastrointestinal tract demonstrated a higher efficacy of PCT in the intestine (pH 6.8) than in the stomach (pH 1.5). Adsorption rates were fast (<2 min) and indicated high capacities (23 µg and 40 µg per 1 mg of PCT at pH 1.5 and pH 6.8, respectively). Allergenically relevant peanut protein concentrations were sorbed in artificial fluids (32 µg/mL by 4 mg/mL of PCT at pH 1.5 and 80.8 µg/mL by 0.25 mg/mL of PCT at pH 6.8) when imitating a daily dose of 2 g of PCT in an average stomach volume of 500 mL. Experiments focusing on the bioavailability of peanut protein attached to PCT revealed sustained sorption at pH 1.5 and only minor desorption at pH 6.8. Accompanied by gluten, peanut proteins showed competing binding characteristics with PCT. This study therefore demonstrates the potential of PCT in binding relevant quantities of peanut allergens during the digestion of peanut-contaminated food.

Structural and Immunological Features of PR-10 Allergens: Focusing on the Major Alder Pollen Allergen Aln g 1.

Today, allergies have become a serious problem. PR-10 proteins are clinically relevant allergens that have the ability to bind hydrophobic ligands, which can significantly increase their allergenicity potential. It has been recently shown that not only the birch pollen allergen Bet v 1 but also the alder pollen allergen Aln g 1, might act as a true sensitizer of the immune system. The current investigation is aimed at the further study of the allergenic and structural features of Aln g 1. By using qPCR, we showed that Aln g 1 was able to upregulate alarmins in epithelial cells, playing an important role in sensitization. With the use of CD-spectroscopy and ELISA assays with the sera of allergic patients, we demonstrated that Aln g 1 did not completely restore its structure after thermal denaturation, which led to a decrease in its IgE-binding capacity. Using site-directed mutagenesis, we revealed that the replacement of two residues (Asp27 and Leu30) in the structure of Aln g 1 led to a decrease in its ability to bind to both IgE from sera of allergic patients and lipid ligands. The obtained data open a prospect for the development of hypoallergenic variants of the major alder allergen Aln g 1 for allergen-specific immunotherapy.

Wheat gluten deamidation: structure, allergenicity and its application in hypoallergenic noodles.

BACKGROUND: Wheat gluten (WG) containing gliadin and glutenin are considered the main allergens in wheat allergy as a result of their glutamine-rich peptides. Deamidation is a viable and efficient approach for protein modifications converting glutamine into glutamic acid, which may have the potential for allergenicity reduction of WG. RESULTS: Deamidation by citric acid was performed to investigate the effects on structure, allergenicity and noodle textural properties of wheat gluten (WG). WG was heated at 100 °C in 1 m citric acid to yield deamidated WG with degrees of deamidation (DD) ranging from DWG-25 (25% DD) to DWG-70 (70% DD). Fourier-transform infrared and intrinsic fluorescence spectroscopy results suggested the unfolding of WG structure during deamidation, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed molecular weight shifts at the 35-63 kDa region, suggesting that the deamidation mainly occurred on low molecular weight glutenin subunits and γ- gliadin of the WG. An enzyme-linked immunosorbent assay of deamidated WG revealed a decrease in absorbance and immunoblotting indicated that the intensities of protein bands at 35-63 kDa decreased, which suggested that deamidation of WG might have caused a greater loss of epitopes than the generation of new epitopes caused by unfolding of WG, and thereby reduction of the immunodominant immunoglobulin E binding capacity, ultimately leading to the decrease in allergenicity. DWG-25 was used in the preparation of recombinant hypoallergenic noodles, and the hardness, elasticity, chewiness and gumminess were improved significantly by the addition of azodicarbonamide. CONCLUSION: The present shows the potential for deamidation of the WG products used in novel hypoallergenic food development. © 2023 Society of Chemical Industry.

Ultrasound-enhanced covalent reaction of gliadin: the inhibition of antigenicity and its potential mechanisms.

BACKGROUND: Wheat proteins can be divided into water/salt-soluble protein (albumin/globulin) and water/salt-insoluble protein (gliadins and glutenins (Glu)) according to solubility. Gliadins (Glia) are one of the major allergens in wheat. The inhibition of Glia antigenicity by conventional processing techniques was not satisfactory. RESULTS: In this study, free radical oxidation was used to induce covalent reactions. The effects of covalent reactions by high-intensity ultrasound (HIU) of different powers was compared. The enhancement of covalent grafting effectiveness between gliadin and (-)-epigallo-catechin 3-gallate (EGCG) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry and Folin-Ciocalteu tests. HIU caused protein deconvolution and disrupted the intrastrand disulfide bonds that maintain the tertiary structure, causing a shift in the side chain structure, as proved by Fourier, fluorescence and Raman spectroscopic analysis. Comparatively, the antigenic response of the conjugates formed in the sonication environment was significantly weaker, while these conjugates were more readily hydrolyzed and less antigenic during simulated gastrointestinal fluid digestion. CONCLUSION: HIU-enhanced free radical oxidation caused further transformation of the spatial structure of Glia, which hid or destroyed the antigenic epitope, effectively inhibiting protein antigenicity. This study widened the application of polyphenol modification in the inhibition of wheat allergens. © 2024 Society of Chemical Industry.

Screening of probiotic Lactobacillus to reduce peanut allergy and with potential anti-allergic activity.

BACKGROUND: Peanut is a significant source of nutrition and a valuable oilseed crop. It is also a serious allergy source, which poses a threat to 1.1% of the population. This study aimed to screen lactic acid bacteria (LAB) with the capacity to alleviate peanut allergenicity and exhibit anti-allergic properties. RESULT: The results show that LAB can make use of substances in peanuts to reduce the pH of peanut milk from 6.603 to 3.593-4.500 by acid production and that it can utilize the protein in peanuts to reduce the allergenic content (especially Ara h 1) and improve biological activity in peanut pulp. The content of Ara h 1 peanut-sensitizing protein was reduced by 74.65% after fermentation. The protein extracted from fermented peanut pulp is more readily digestible by gastrointestinal juices. The inhibitory activity assay of hyaluronidase (an enzyme with strong correlation to allergy) increased from 46.65% to a maximum of 90.57% to reveal that LAB fermentation of peanut pulp exhibited a robust anti-allergic response. CONCLUSION: The strains identified in this study exhibited the ability to mitigate peanut allergenicity partially and to possess potential anti-allergic properties. Lactobacillus plantarum P1 and Lactobacillus salivarius C24 were identified as the most promising strains and were selected for further research. © 2023 Society of Chemical Industry.

Effect of transglutaminase crosslinking combined with lactic fermentation on the potential allergenicity and conformational structure of soy protein.

BACKGROUND: Food allergies are a growing concern worldwide, with soy proteins being important allergens that are widely used in various food products. This study investigated the potential of transglutaminase (TGase) and lactic acid bacteria (LAB) treatments to modify the allergenicity and structural properties of soy protein isolate (SPI), aiming to develop safer soy-based food products. RESULTS: Treatment with TGase, LAB or their combination significantly reduced the antibody reactivity of ß-conglycinin and the immunoglobulin E (IgE) binding capacity of soy protein, indicating a decrease in allergenicity. TGase treatment led to the formation of high-molecular-weight aggregates, suggesting protein crosslinking, while LAB treatment resulted in partial protein hydrolysis. These structural changes were confirmed by Fourier transform infrared spectroscopy, which showed a decrease in ß-sheet content and an increase in random coil and ß-turn contents. In addition, changes in intrinsic fluorescence and ultraviolet spectroscopy were also observed. The alterations in protein interaction and the reduction in free sulfhydryl groups highlighted the extensive structural modifications induced by these treatments. CONCLUSION: The synergistic application of TGase and LAB treatments effectively reduced the allergenicity of SPI through significant structural modifications. This approach not only diminished antibody reactivity of ß-conglycinin and IgE binding capacity of soy protein but also altered the protein's primary, secondary and tertiary structures, suggesting a comprehensive alteration of SPI's allergenic potential. These findings provide a promising strategy for mitigating food allergy concerns and lay the foundation for future research on food-processing techniques aimed at allergen reduction. © 2024 Society of Chemical Industry.

Quantifying allergenic proteins using antibody-based methods or liquid chromatography-mass spectrometry/mass spectrometry: A review about the influence of food matrix, extraction, and sample preparation.

Accurate quantification of allergens in food is crucial for ensuring consumer safety. Pretreatment steps directly affect accuracy and efficiency of allergen quantification. We systematically reviewed the latest advances in pretreatment steps for antibody-based methods and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) protein quantification methods in food. For antibody-based methods, the effects induced by food matrix like decreased allergen solubility, epitope masking, and nonspecific binding are of the upmost importance. To mitigate interference from the matrix, effective and proper extraction can be used to obtain the target allergens with a high protein concentration and necessary epitope exposure. Removal of interfering substances, extraction systems (buffers and additives), assistive technologies, and commercial kits were discussed. About LC-MS/MS quantification, the preparation of the target peptides is the crucial step that significantly affects the efficiency and results obtained from the MS detector. The advantages and limitations of each method for pre-purification, enzymatic digestion, and peptide desalting were compared. Additionally, the application characteristics of microfluidic-based pretreatment devices were illustrated to improve the convenience and efficiency of quantification. A promising research direction is the targeted development of pretreatment methods for complex food matrices, such as lipid-based and carbohydrate-based matrices.

Animal-derived food allergen: A review on the available crystal structure and new insights into structural epitope.

Immunoglobulin E (IgE)-mediated food allergy is a rapidly growing public health problem. The interaction between allergens and IgE is at the core of the allergic response. One of the best ways to understand this interaction is through structural characterization. This review focuses on animal-derived food allergens, overviews allergen structures determined by X-ray crystallography, presents an update on IgE conformational epitopes, and explores the structural features of these epitopes. The structural determinants of allergenicity and cross-reactivity are also discussed. Animal-derived food allergens are classified into limited protein families according to structural features, with the calcium-binding protein and actin-binding protein families dominating. Progress in epitope characterization has provided useful information on the structural properties of the IgE recognition region. The data reveals that epitopes are located in relatively protruding areas with negative surface electrostatic potential. Ligand binding and disulfide bonds are two intrinsic characteristics that influence protein structure and impact allergenicity. Shared structures, local motifs, and shared epitopes are factors that lead to cross-reactivity. The structural properties of epitope regions and structural determinants of allergenicity and cross-reactivity may provide directions for the prevention, diagnosis, and treatment of food allergies. Experimentally determined structure, especially that of antigen-antibody complexes, remains limited, and the identification of epitopes continues to be a bottleneck in the study of animal-derived food allergens. A combination of traditional immunological techniques and emerging bioinformatics technology will revolutionize how protein interactions are characterized.