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In this chapter, the analytical workflow typically used for the development and validation of an analytical method tailored to food allergen detection and quantification is presented. The main steps defining the workflow are herein described and commented with specific notes about the critical issues that can be faced and common solutions to be adopted. References to guidelines and/or recommendation available from official bodies, as well as main papers from international consortia operating on the specific research field, are also reported, whenever possible. As such, this chapter may represent a practical guide to drive method development in the standardization of analytical methods for food allergen detection., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Peptide marker identification is an important step in development of a mass spectrometry method for multiple allergen detection, since specificity, robustness and sensitivity of the overall analytical method will depend on the reliability of the proteotypic peptides. As part of the development of a multi-analyte reference method, discovery analysis of two incurred food matrices has been undertaken to select the most reliable peptide markers. Six allergenic ingredients (milk, egg, peanut, soybean, hazelnut, and almond) were incurred into either chocolate or broth powder matrix. Different conditions of protein extraction and purification were tested and the tryptic peptide pools were analysed by untargeted high resolution tandem mass spectrometry and the resulting fragmentation spectra were processed via a commercial software for sequence identification. The analysis performed on incurred foods provides both a prototype effective and straightforward sample preparation protocol and delivers reliable peptides to be included in a standardized selected reaction monitoring method., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The goji berry ( Lycium barbarum L.) (GB) is gaining increasing attention with high consumption worldwide due to its exceptional nutritional value and medicinal benefits displayed in humans. Beyond their beneficial properties, GBs contain renowned allergenic proteins, and therefore deserve inclusion among the allergenic foods capable of inducing allergic reactions in sensitive consumers. GB allergy has been frequently linked to the panallergen lipid transfer protein (LTP), especially across the population of the Mediterranean area. Methods: In this study, we investigated the protein profile of GBs focusing on the most reactive proteins against immunoglobulins E (IgE) of allergic patients' sera, as ascertained by immunoblot experiments. The protein spots displaying a clear reaction were excised, in-gel digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by data searching against a restricted database for a reliable protein identification. Results: According to our data, three main spots were identified in GB extract as IgE binding proteins after immunoblot analysis. Some major proteins were identified and the three proteins that provided the highest reactivity were putatively attributed to vicilin and legumin proteins followed by a protein matching with 11S globulin belonging to the cupin superfamily. Finally, the whole GB protein extract was also submitted to bottom-up proteomics followed by a software-based database (DB) screening and a more exhaustive list of GB proteins was compiled.

Food allergy is a large and growing public health problem in many areas of the world. The prevalence of food allergy has increased in the last decades in a very significant way in many world regions, particularly in developed countries. In that respect, the research field of food allergy has experienced an extensive growth and very relevant progress has been made in recent years regarding the characterization of food allergens, the study of their immunological properties, and their detection in food sources. Furthermore, food labeling policies have also been improved decidedly in recent years. For that immense progress made, it is about time to review the latest progress in the field of food allergy. In this review, we intend to carry out an extensive and profound overview regarding the latest scientific advances and knowledge in the field of food allergen detection, characterization, and in the study of the effects of food processing on the physico-chemical properties of food allergens. The advances in food labeling policies, and methodologies for the characterization of food allergens are also thoroughly reviewed in the present overview., Competing Interests: Conflict of interest The authors declare no conflict of interest., (© 2020 Elsevier Inc. All rights reserved.)

Peanuts are a source of proteins and fats but they are also considered a harmful food for individuals who are allergic to them due to their ability to trigger severe and life-threatening reactions. Strict avoidance of peanuts is the most effective means to prevent the development of an allergic reaction. Physical or chemical strategies employing autoclaving can represent a valid alternative to produce a final food with a decreased allergenic power as in the case of peanuts. Thermal processing might induce protein modifications in foods and affect protein digestibility or absorption of nutrients across the intestinal mucosa. Besides, the type of processing could also alter food protein allergenicity thus influencing the interplay with the biological system at the gut level. In this paper, we investigated the influence of autoclaving based treatments on the proliferation of epithelial cells at the intestinal level. Extractable proteins of raw and autoclaved peanuts were analysed by SDS-PAGE and untargeted LC-high resolution-MS/MS to investigate the peptide composition. Our findings show that when raw peanuts were assayed on Caco 2 cell lines, an antiproliferative effect was observed. By contrast, peanuts subjected to hydration and autoclaving did not show an inhibition of proliferation on Caco-2 cells. In parallel, extensive fragmentation induced by autoclaving treatments on the original peanut proteins was also recorded by LC-MS/MS analysis with a consequent increase in the number of peptides detected. These results indicate that the processing applied to peanuts can have an influence on both the nutritional and allergological sides, and more investigations will be required on this issue to understand the alteration of inflammatory mediators induced by the treatment applied.

In recent years, mass spectrometry (MS) has been establishing its role in the development of analytical methods for multiple allergen detection, but most analyses are being carried out on low-resolution mass spectrometers such as triple quadrupole or ion traps. In this investigation, performance provided by a high resolution (HR) hybrid quadrupole-Orbitrap™ MS platform for the multiple allergens detection in processed food matrix is presented. In particular, three different acquisition modes were compared: full-MS, targeted-selected ion monitoring with data-dependent fragmentation (t-SIM/dd2), and parallel reaction monitoring. In order to challenge the HR-MS platform, the sample preparation was kept as simple as possible, limited to a 30-min ultrasound-aided protein extraction followed by clean-up with disposable size exclusion cartridges. Selected peptide markers tracing for five allergenic ingredients namely skim milk, whole egg, soy flour, ground hazelnut, and ground peanut were monitored in home-made cookies chosen as model processed matrix. Timed t-SIM/dd2 was found the best choice as a good compromise between sensitivity and accuracy, accomplishing the detection of 17 peptides originating from the five allergens in the same run. The optimized method was validated in-house through the evaluation of matrix and processing effects, recoveries, and precision. The selected quantitative markers for each allergenic ingredient provided quantification of 60-100 μg ingred /g allergenic ingredient/matrix in incurred cookies.

Peanut allergy is one of the most widespread types of food allergies especially affecting developed countries. To reduce the risk of triggering allergic reactions, several technological strategies have been devised to modify or remove allergens from foods. Herein we investigated the combination of high temperature and pressure on the modulation of peanuts immunoreactivity after simulated gastro-duodenal digestion. Extractable proteins of raw and autoclaved peanuts were separated on SDS-PAGE and immunogenicity was assessed by ELISA and Western Blot analyses. Proteins surviving the heat treatment and reacting towards allergic patients' sera were analysed and attributed to Ara h 3 and Ara h 1 proteins by untargeted LC-high resolution-MS/MS. A progressive reduction in the intensity of the major allergen proteins was also highlighted in the protein fraction extracted from autoclaved peanuts, with a total disappearance of the high molecular allergens when samples were preliminary exposed to 2 h hydration although the lower molecular weight fraction was not investigated in the present work. Furthermore, raw and processed peanuts underwent simulated digestion experiments and the IgE binding was assessed by using allergic patients' sera. The persistence of an immunoreactive band was displayed around 20 kDa. In conclusion, the synergistic effects of heat and pressure played a pivotal role in the disappearance of the major peanut allergens also contributing to the significant alteration of the final immunoreactivity. In addition, the surviving of allergenic determinants in peanuts after gastrointestinal breakdown provides more insights on the fate of allergenic proteins after autoclaving treatments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Allergenic ingredients in pre-packaged foods are regulated by EU legislation mandating their inclusion on labels. In order to protect allergic consumers, sensitive analytical methods are required for detect allergen traces in different food products. As a follow-up to our previous investigations, an optimized, sensitive, label-free LC-MS/MS method for multiplex detection of five allergenic ingredients in a processed food matrix is proposed. A cookie base was chosen as a complex food matrix and home-made cookies incurred with whole egg, skimmed milk, soy flour, ground hazelnut and ground peanut were prepared at laboratory scale. In order to improve the analytical workflow both protein extraction and purification protocols were optimized and finally a sensitive streamlined SRM based analytical method for allergens detection in incurred cookies was devised. The effect of baking on the detection of selected markers was also investigated., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

There is a raising demand for sensitive and high throughput MS based methods for screening purposes especially tailored to the detection of allergen contaminants in different food commodities. A challenging issue is represented by complex food matrices where the antibody-based kits commercially available might encounter objective limitations consequently to epitope masking phenomena due to a multitude of interfering compounds arising from the matrix. The performance of a method duly optimized for the extraction and simultaneous detection of soy, egg and milk allergens in a cookie food matrix by microHPLC-ESI-MS/MS, is herein reported. Thanks to the innovative configuration and the versatility shown by the dual cell linear ion trap MS used, the most intense and reliable peptide markers were first identified by untargeted survey experiment, and subsequently employed to design an ad hoc multi-target SRM method, based on the most intense transitions recorded for each selected precursor peptide. A sample extraction and purification protocol was optimized also including an additional step based on sonication, which resulted in a considerable improvement in the detection of milk allergen peptides. Data Dependent™ Acquisition scheme allowed to fill out a tentative list of potential peptide markers, which were further filtered upon fulfilling specific requirements. A total of eleven peptides were monitored simultaneously for confirmation purposes of each allergenic contaminant and the two most sensitive peptide markers/protein were selected in order to retrieve quantitative information. Relevant LODs were found to range from 0.1μg/g for milk to 0.3μg/g for egg and 2μg/g for soy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Rationale: A method based on High-Resolution Mass Spectrometry was developed for the simultaneous determination of fining agents containing potentially allergenic milk (casein) and egg-white (lysozyme and ovalbumin) proteins, added to commercial white wines at sub-ppm levels. Selected tryptic peptides were used as quantitative markers. An evaluation of protein digestion yields was also performed by implementing the (15)N-valine-labelled analogues of the best peptide markers identified for αS1 -casein and ovalbumin., Methods: The method was based on the combination of ultrafiltration (UF) of protein-containing wines, tryptic digestion of the dialyzed wine extracts and liquid chromatography/high resolution mass spectrometry (LC/HRMS) analysis of tryptic digests. Peptides providing the most intense electrospray ionization (ESI)-MS response were chosen as quantitative markers of the proteins under investigation., Results: Six-point calibrations were performed by adding caseinate and egg-white powder in the concentration range between 0.25 and 10 µg/mL, to an allergen-free white wine. The following three peptide markers, LTEWTSSNVMEER, GGLEPINFQTAADQAR and ELINSWVESQTNGIIR, were highlighted as best markers for ovalbumin, while GTDVQAWIR and NTDGSTDYGILQINSR for lysozyme and YLGYLEQLLR, GPFPIIV and FFVAPFPEVFGK for caseinate. Limits of detection (LODs) ranged from 0.4 to 1.1 µg/mL., Conclusions: The developed method is suited for assessing the contemporary presence of allergenic milk and egg proteins characterizing egg white and caseinate, fining agents typically employed for wine clarification. The LODs of the method enable the detection of sub-ppm concentrations of residual fining agents, that could represent a potential risk for allergic consumers., (Copyright © 2013 John Wiley & Sons, Ltd.)

Due to the growing global incidence of allergy to nuts and peanuts, the need for better protection of consumers sensitive to those products is constantly increasing. The best strategy to defend them against adverse immunological reactions still remains the total removal of those products from their diet. However, nuts and peanuts traces can also be hidden in other food products, especially processed ones, such as bakery products, because of cross-contamination occurring during production. Precautionary labelling is often adopted by producers to warn allergic consumers, usually without any evaluation of the actual risk, which would require a careful quantification of nuts/peanuts traces. In this paper, the development of a multi-target method based on liquid chromatography-tandem high resolution mass spectrometry (LC-MS, MS/MS), able to detect traces of five nuts species (almonds, hazelnuts, walnuts, cashews and pistachios) and of peanuts in an in-house incurred bakery product (cookie) through a single analysis is described. Specifically, allergenic proteins of the six ingredients were used as the analytical targets, and the LC-MS responses of selected peptides resulting from their tryptic digestion, after extraction from the bakery product matrix, were exploited for quantification, following a bottom-up approach typical of proteomics. As a result, nuts/peanuts could be detected/quantified down to mg·kg−1 levels in the model cookie, thus opening interesting perspectives for the quantification of hidden nuts/peanuts in bakery products and, consequently, for a more rational use of precautionary labelling. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS) represents an essential tool in proteomics studies, in the last years also exploited for monitoring allergens contamination in food products. Milk and egg are renowned allergens often used as fining agents to promote clarification of wines, therefore any residual amount in the end-products could represent a menace for allergic individuals. In view of this, it is of paramount importance to have at disposal sensitive analytical methods able to detect traces of milk and egg allergens in food. In this work we describe the upgrade and the optimization of an analytical workflow based on the use of a pre-enrichment column coupled with HPLC separation and MS/MS detection for the selective and sensitive detection of milk and egg allergens in white wine. Two different sample pre-treatments based on the use of mass cut-off filters or size exclusion cartridges were evaluated and compared, before tryptic digestion and LC-SRM-MS/MS analysis of the resulting peptides mixture. The devised UF based method coupled with peptide on-line pre-enrichment enabled to reach the lowest LODs down at 0.036 μg/mL and 0.05 μg/mL for egg and milk allergens respectively, proving to be the most sensitive strategy for monitoring allergens contamination in wine. [ABSTRACT FROM AUTHOR]

Peanut represents one of the most harmful allergenic foods capable of triggering severe and sometimes lethal reactions in allergic consumers upon ingestion of even small amounts. Several proteins capable of inducing allergic reactions that have been recognised by patients’ IgE antibodies have been identified from this nut source. Methods mainly based on ELISA assays have been developed in order to detect peanuts in several food commodities. In addition LC-MS/MS methods based on different mass analysers have also been devised for tracing peanut contamination in different foods achieving low limits of detection. The applicability of a benchtop high-resolution Exactive™ mass spectrometer has never been investigated for the rapid screening of peanut contamination in complex food matrices like mixtures of nuts. We report in this paper the design of suitable peanut markers and the development of an high-resolution Orbitrap™ mass spectrometer-based method for peanut detection in a mixture of nuts species. With this aim, different types of samples were prepared: (1) nuts-based powder made up of a mixture of hazelnuts, pistachios, almonds and walnuts; and (2) nuts powder fortified with peanuts. Different levels of fortifications were produced and the applicability of the method was tested. Finally, a subset of six peptides fulfilling specific analytical requirements was chosen to check the suitability of the method tailored to the detection of peanuts in nuts-based products, and two of them, peptides VYD and WLG, were selected as quantitative markers. The method proved to be a suitable screening tool to assess the presence of traces of peanuts in other tree nuts with a limit of detection as low as 4 µg of peanuts proteins or 26 µg of peanuts in 1 g of matrix. [ABSTRACT FROM PUBLISHER]

Food allergen research has made giant steps in the last years thanks to the features offered by the latest technology of mass analyzers placed on the market allowing multiplex sensitive detection of proteins. Potentials and features of two mass analyzers namely a linear ion trap capable of performing a data dependent or selected reaction monitoring analysis and an OrbitrapTM stand-alone MS enabling a broadband fragmentation without mass selection at highest mass resolving power are herein described and applied to the multiplex screening of allergens in a type of wine chosen as a reference matrix. Quantitative and confirmative capabilities of both platforms were assessed on the specific case study, the multiple detection of egg and milk -related proteins, typically employed in white wines as fining agents. Commercial bioinformatic tools used for a quick allergen identification will be also discussed. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Consumption of tree nuts and peanuts has considerably increased over the last decades due to their nutritional composition and the content of beneficial compounds. On the other hand, such widespread consumption worldwide has also generated a growing incidence of allergy in the sensitive population. Allergy to nuts and peanuts represents a global relevant problem, especially due to the risk of the ingestion of hidden allergens as a result of cross-contamination between production lines at industrial level occurring during food manufacturing. The present review provides insights on peanuts, almonds, and four nut allergens—namely hazelnuts, walnuts, cashew, and pistachios—that are likely to cross-contaminate different food commodities. The paper aims at covering both the biochemical aspect linked to the identified allergenic proteins for each allergen category and the different methodological approaches developed for allergens detection and identification. Attention has been also paid to mass spectrometry methods and to current efforts of the scientific community to identify a harmonized approach for allergens quantification through the detection of allergen markers. [ABSTRACT FROM AUTHOR]

Hazelnut is a widespread nut species, especially present in Europe, that can be consumed raw or roasted thanks to its pleasant taste and nutritional properties. In addition to renowned beneficial properties hazelnuts contain several proteins capable of inducing food allergy in sensitized individuals, including Cor a 2 (a profilin), Cor a 8 (a lipid transfer protein), Cor a 9 (an 11S seed storage globulin, legumin-like), and Cor a 11 (a 7S seed storage globulin, vicilin-like). In the present paper we investigated the effectiveness of autoclave-based treatments in decreasing the allergic potential of hazelnut as assessed by submitting the treated material to an in vivo skin prick test and an in vitro immunoblot analysis, with sera of allergic individuals exposed to the treated food material. This preliminary analysis showed that autoclave treatment preceded by hydration and/or followed by drying seems to be a promising approach and appears to be effective in reducing the allergenicity of hazelnuts in most patients, probably due to the denaturation of most major and minor allergenic proteins. This work opens up the opportunity to produce hypoallergenic hazelnut derivatives that can be tolerated by allergic subjects. [ABSTRACT FROM AUTHOR]

The prevalence of food allergy has increased over the last decades and consequently the food labeling policies have improved over the time in different countries to regulate allergen presence in foods. In particular, Reg 1169 in EU mandates the labelling of 14 allergens whenever intentionally added to foods, but the inadvertent contamination by allergens still remains an uncovered topic. In order to warn consumers on the risk of cross-contamination occurring in certain categories of foods, a precautionary allergen labelling system has been put in place by food industries on a voluntary basis. In order to reduce the overuse of precautionary allergen labelling (PAL), reference doses and action limits have been proposed by the Voluntary Incidental Trace Allergen Labelling VITAL project representing a guide in this jeopardizing scenario. Development of sensitive and reliable mass spectrometry methods are therefore of paramount importance in this regard to check the contamination levels in foods. In this paper we describe the development of a time-managed multiple reaction monitoring (MRM) method based on a triple quadrupole platform for milk and egg quantification in processed food. The method was in house validated and allowed to achieve levels of proteins lower than 0.2 mg of total milk and egg proteins, respectively, in cookies, challenging the doses recommended by VITAL. The method was finally applied to cookies labeled as milk and egg-free. This method could represent, in perspective, a promising tool to be implemented along the food chain to detect even tiny amounts of allergens contaminating food commodities. [ABSTRACT FROM AUTHOR]