Rosa Pilolli

Chiara Nitride

Nathalie Gillard

Anne-Catherine Huet

Christof Van Poucke

Marc De Loose

Olivier Tranquet

Colette Larre

Karine Adel-Patient

Hervé Bernard

Collette Larré

E N Clare Mills

Linda Monaci
email: linda.monaci@ispa.cnr.it

Critical review on proteotypic peptide marker tracing for six allergenic ingredients in incurred foods by mass spectrometry

Keywords: food allergens, mass spectrometry, peptide markers, ThRAll, incurred foods

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

IgE-mediated food allergies are steadily on the rise representing a huge health concern worldwide. Although firm prevalence data are lacking, there are extensive investigations suggesting that food allergies have increased and rates as high as approximately 10 % have been documented, with main prevalence in industrialized countries, and in children compared to adults [START_REF] Scott | Food allergy: A review and update on epidemiology, pathogenesis, diagnosis, prevention, and management[END_REF]. There are many manifestations of food allergy with different severities, and individual investigations rely on specific study populations, specific foods, and different methodologies, thus impairing the determination of undoubted statistics. Moreover, further constrains in obtaining solid prevalence data, arise from geographic variations, diet exposure effects, differences according to age, race, and ethnicity, and myriad other factors. Like all chronic disease, onset of food allergy is influenced by genetics, environment and genome-environment interactions including epigenetics effects. Its current management is based on allergen containing food avoidance. Food labelling legislative provisions are available in several countries and require a detailed declaration of allergen inclusion in foods, with number and types of priority allergens differing among countries and depending on specific dietary habits and allergy prevalence. On the other hand, a precautionary labeling system is also widespread, representing a safety measure put in place from industries to protect from any accidental cross-contamination likely to occur in foods expected to be allergen-free and that can take place at whatever stage along the food chain. All this raised the urgent need to develop reliable methodologies to trace allergens in foods with the highest confidence. Mass spectrometry (MS) techniques have been widely exploited in the last ten years to meet this goal and were specifically applied to food allergen detection, and to the food allergen identification and characterization, as well. Despite the need for expensive equipment and trained personnel, the chance to provide multiplexing and unequivocal allergen identification accounts for the overall strength of the MS based approaches compared to previously established methods, such as immunoassays and DNA based methods. Nonetheless some gaps in the development of MS-based quantitative methods still need to be addressed. The review paper authored by [START_REF] Monaci | Comprehensive overview and recent advances in proteomics MS based methods for food allergens analysis[END_REF] provides a comprehensive overview of the MS based methods developed so far for allergen detection and also highlights the need of harmonization in method development and validation.

In this frame, the European project ThRAll (Thresholds and Reference method for Allergen detection method) titled "Detection and quantification of allergens in foods and minimum eliciting doses in food allergic individuals", funded by the European Food Safety Agency will actively contribute to the advancement in harmonization of MS-based method for food allergen detection by developing a prototype quantitative reference method for the multiple detection of food allergens in standardized incurred food matrices [START_REF] Mills | Detection and Quantification of Allergens in Foods and Minimum Eliciting Doses in Food-Allergic Individuals (ThRAll)[END_REF]. Five main food ingredients causing severe IgE-mediated reactions have been prioritized in light of their occurrence in food recalls [START_REF] Turner | Increase in anaphylaxis-related hospitalizations but no increase in fatalities: an analysis of United Kingdom national anaphylaxis data, 1992-2012[END_REF][START_REF] Worm | First European data from the network of severe allergic reactions (NORA)[END_REF]Bucchini et al., 2016). Milk, soybean, tree nuts, egg, and peanuts were selected as responsible for most alerts notified on the RASFF (Rapid Alert System for Food and Feed) portal for undeclared presence with serious risk decision. A recent notice from the European Commission (Commission Notice of 13.7.2017) relating to the provision of information on substances or products causing allergies or intolerances as listed in Annex II of Regulation (EU) No 1169/2011 provides specifications about how the Annex II of Regulation (EU) No 1169/2011 should be implemented. In particular, as for milk and egg ingredients the notice clarifies that the legislation should be applied to milk from all farmed animals and egg from all farmed birds. Given the timing of the ThRAll project, and the urgent demand for harmonization of current analytical methods, the Consortium agreed to prioritize cow's milk and hen's egg allergens, as first targets, also in light of their main role in the primary production. Indeed according to recent statistical data (FaoStat, 2017), cow's milk production represented in 2017 the 97% of total milk production in Europe and the 81% of the production worldwide. Similarly, in the same year the hen's eggs accounted for the 99% of total egg production in Europe and for the 82% worldwide. Whether the developed approach would be applicable to all farmed animals/birds will be specified depending on the final validated analytical markers. As for tree nuts allergens, two representative ingredients have been selected, hazelnut and almond, which are widely used in food manufacturing and represent foods of public health importance in Europe, causing severe reactions [START_REF] Worm | First European data from the network of severe allergic reactions (NORA)[END_REF].

The allergenic ingredients will need to be incorporated into the food before being processed to mimic as closely as possible the manufacturing process. Two model matrices will need to be produced ad hoc in a food pilot plant, namely a chocolate bar and a broth powder. These latter are the representative food commodities responsible for recalls, namely baked goods, confectionary products and complex multiphase foods and pose specific challenges in MS based analytical detection. Both matrices were selected because they are extremely complex, with different features, having chocolate high contents of fat and polyphenols whilst broth powder is an extensive processed food (cooking, boiling and drying), enriched with proteins.

One of first tasks scheduled in the ThRAll project (Objective 1) concerns the identification of protein and peptide markers for the targeted allergenic ingredients. As a fact, markers identification is one of the most important steps in the method development. Different workflows are available to accomplish this objective and they have been reviewed in a recent paper authored by [START_REF] Downs | Target selection strategies for LC-MS/MS food allergen methods[END_REF] All issues besetting such choice including, various ingredients formulations, extraction yield in thermally processed foods, and incomplete genomic and proteomic sequence information, as well as all common issues associated with MS-based protein quantitation were critically discussed. The main point raised by Downs & Johnson was that the target selection cannot be accounted by in-silico only approach. Methodological details, such as matrix composition, protein extraction and digestion, should be taken into consideration for marker selection, affecting both the specificity and sensitivity of the final analytical method.

The assessment on a model matrix is required firstly for specificity issue because interfering peaks from the matrix background may cause false-positives; in addition the processing degree of the investigated food matrix may also differently affect the target protein/peptide detection resulting in false-negative which can pose safety risk for the allergic consumer. None of these issues can be tackled by the solely in-silico based approach, thus the need for an empirical validation of the candidate peptide markers in incurred foods [START_REF] Monaci | Comprehensive overview and recent advances in proteomics MS based methods for food allergens analysis[END_REF].

An alternative approach could rely on the comprehensive assessment of the allergen detection literature, supported by the experimental evidence that if the same peptides for given allergenic proteins have been detected in independent studies and in multiple matrices, they can likely be standardized [START_REF] Croote | Food allergen detection by mass spectrometry: the role of systems biology[END_REF]. In order to promote this approach, an open-source repository of discovery and targeted MS data on allergen detection and quantitation has been developed and made available as public bioinformatic tool (Allergen Peptide Browser, 2018).

The database has been structured around proteins recognized by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) as food allergens.

After a proper screening of the different available options, the partners of ThRAll consortium agreed on undertaking peptide selection by applying a dual approach with different time scale: (i) critical evaluation of the peptide markers already reported in the existing literature; (ii) validation of the candidate peptide list by discovery analysis on the specific incurred matrices under investigation (chocolate bars and broth powder). Herein, we will describe the results of the first point, namely the comprehensive evaluation of the literature, providing a thorough discussion about the application of general acceptance criteria for harmonization of the markers selection step. The goal of this work is to draw a preliminary list of reliable peptide markers, which will be validated later on by untargeted HR-MS/MS analysis of incurred matrices, representing the bases for the selected reaction monitoring (SRM) based reference method under development within the ThRAll project.

Literature review based marker peptide selection

The analytical workflow displayed in Fig. 1 was designed to provide the comprehensive evaluation of the current literature. It consists of five main steps aimed at the critical evaluation of the information gathered from previous works and the proper selection of reported signature peptides according to specific criteria. We collected all information about the six allergens selected as targets in the ThRAll project (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) (step 1). Afterwards, the list of reported signature peptides have been evaluated and filtered according to specific features, namely sequence length, matrix similarity with the ThRAll project, kind of investigation (discovery only or targeted analysis) and occurrence of amino acid residues prone to endogenous and exogenous modifications (step 2). The third step of the workflow consisted in retrieving all the information available for the relevant allergenic proteins (relative abundance, post-translational modification sites, protein isoforms and variants), from different informatics sources detailed in Figure 1. At step 4 peptide specificity was assessed by sequence alignment with other species searching not only for exact matches of tryptic peptides (performed for all candidate markers of the six allergenic ingredients) but also disclosing potential interference from sequence homology with single amino acid mismatches (performed only on the four plant-derived allergenic ingredients). Finally as last step of the analytical workflow, for the refined peptide list we collected experimental details about the MS-based detection, such as the instrumental platform, the precursor ions, and the selected transitions. Indeed, the peptide ionization efficiency and reproducibility, as well as the relative intensity of fragmentation pattern might be affected also by instrumental parameters which need to be assessed case-by-case. For the comprehensive assessment of the allergen detection literature (step 1 in Fig. 1) we exploited the Allergen Peptide Browser (APB) database available online, and collected all information about the six allergens selected for the ThRAll project. The gathered information also integrated data fed by other studies based on MS/MS analysis with either ESI or MALDI ionization, encompassing the most recent publications not itemized yet on APB. A total of 89 papers were reviewed, published in the last twenty-five years, which resulted in an exhaustive list of tryptic peptides for milk (total 42 peptides), egg (total 62 peptides), peanut (total 50 peptides) and soybean (total 146 peptides), and a shorter, but still reasonable list for hazelnut (total 26 peptides) and almond (total 29 peptides) allergens (see Table S1). In the second step the full list was narrowed down according to specific constraints.

A range of 7-20 amino acids (AA) in length was applied as good compromise between peptide specificity, ionization yield and reproducibility. About 84% of reported peptides were included in this range. Afterwards, we searched for matrix similarity with the ThRAll project, and marker peptides that were never validated in either chocolate or highly processed incurred matrices were rejected. The resulting peptides list is displayed in Fig. 2 and Fig. 3 (and itemized in Table S2) together with details about the total number of citing papers per each peptide, and the kind of investigation (targeted vs discovery approaches), marked with different color (orange and blue, respectively).

The marker selection should comply with specific requirements that value the quantitative information retrieved. Indeed, the allergen quantification depends on the concentration of peptide markers, based on the assumption that the peptide is released completely from the original protein with a specific stoichiometric ratio. Any unpredictable distribution of the released peptide into different precursor ions, for example due to amino acid modification or multiple ionization states, should be traced and susceptible peptides should be excluded, in case of low reproducibility. Concerning the exclusion of signature peptides containing labile amino acids prone to modifications, it deserves to be noted that the number of potential endogenous and exogenous post-translational modifications is huge, but their occurrence is highly variable; therefore, it is not a realistic goal neither to evaluate nor to exclude them all strictly, rather an empirical validation of the specific case study should be preferable. In this investigation, we focused on the occurrence of specific amino acids of major concern, such as methionine, asparagine, glutamine, and cysteine, excluding only peptide sequences containing methionine residues prone to processing-induce oxidation, and asparagine-glycine motifs prone to deamidation [START_REF] Li | Effects of acidic N+1 residues on asparagine deamidation rates in solution and in the solid state[END_REF]Li et al., 2006). We deemed important for the robustness of the analytical method to avoid M containing peptides as analytical targets, because such residue would be highly prone to oxidation, in thermally processed matrices, in barely predictable proportions. The NG motifs could be subjected to spontaneous deamidation in-vitro, however, in case of need the asparagine deamidation might be forced in order to convert all asparagine residues into aspartic acid. As for cysteine residues, most digestion protocols included a reduction/alkylation step (mainly based on dithiothreitol and iodoacetamide), preliminary to actual enzymatic proteolysis, which is specifically designed to break disulfide bridges and block irreversibly the cysteine residues by carbamidomethylation. Common sense is to consider this modification as complete however, in the perspective to avoid source of uncertainty, C containing peptides should be preferentially excluded unless the reliability of the peptide is duly assessed experimentally.

As third step of the analytical workflow reported in Fig. 1, starting from the refined list of peptide markers, we sought for additional information available on the target proteins, which could affect the detection sensitivity and reliability, such as relative abundance, natural post-translational modification (PTM), protein isoforms and variants, gathering data from different sources (either scientific publications or on-line databases, i.e. Uniprot, Allergome, WHO/IUIS).

Peptide specificity was assessed by BLAST search against the most common protein databases disclosing also potential detection interferences in plant-derived proteins (single amino acid mismatches) (step 4). The APB Database queried for this review of peptide markers, already provided information about sequence specificity, retrieved by means of BLAST 2.2.31+ (BLAST DB, Sep 20, 2016). Only hits with 100% identity and no gaps were retained for a given peptide, and the following constrains were applied to exclude hits: (i) bacterial super kingdom;

(ii) absent species name or kingdom; (iii) species / common name entry containing: 'synthetic construct' or 'vector'; (iv) title containing 'partial' or 'fragment'. Since it was not clear whether they considered or not the site specific trypsin cleavage, we also double-checked the specificity for all the six allergenic ingredients by means of the Protein Prospector tool developed by the University of California San Francisco (http://prospector.ucsf.edu/prospector/mshome.htm, accessed on October 24th 2018). In particular, the MS-Homology option searching for 100% identity of tryptic peptides within the main databases, NCBInr.2013.6.17 and UniProtKB.2017.11.01, was applied. In addition, for plant-derived allergens, protein homology among different taxonomies needs to be considered very carefully. Besides the previously discussed full matches (100% identity), in low-resolution MS detection potential interferences might raise also under particular conditions, from peptides with single amino acid mismatches. Therefore, further BLAST search against non-redundant protein database was carried out (access in July 2018), seeking for single amino acid substitutions, which could result in differences of peptide molecular weight within ±1 Da (for example, I/L, D/N, E/Q). These kind of substitutions might generate interfering precursor ions isolated and activated at least partially in the set m/z window, and might provide several transitions, which are not distinguishable from the targeted peptides. Finally, as step 5 of the workflow we retrieved experimental details about the MS platform, the isolated ions and the monitored transitions. As a fact, most tryptic peptides feature favorable ionization properties, which provide high ionization efficiency and typically result in either double or triple charged ions due to the occurrence of basic amino acid residues. However, some peptide sequences may present multiple ionization routes resulting in a loss of sensitivity due to the distribution of the total amount of released peptide in different precursor ions, each characterized by a specific fragmentation pattern. Since we use as a calibrant the synthetic natural version of the peptide and use as an anchor the ration measurement to the isotopic labelled version of the peptide, the distribution among different charge state is accounted and thereof can be considered negligible. From the quantitative point of view, by selecting the most intense precursor ions loss of sensitivity can be limited. Besides the amino acid sequence, several experimental features may affect this ionization behavior, e.g. the electrospray source parameters, the MS platform, the composition of the food matrix, dragging competing species to the ionization step. Therefore it is crucial that the theoretical selection of candidate markers, based either on the in-silico approach or on the literature review approach should be confirmed by experimental validation on the specific MS platform and on the main matrices of interest.

In the sections below, we present the results of the analytical workflow reviewing all information collected for the six allergens of interest.

Proteins and peptide markers reported for cow's milk and hen's egg

The inclusion of milk from domesticated mammalian animals, mainly cattle, buffalo, sheep, goat and camel, boasts a very long tradition in the human diet. According to the FAO data on primary livestock production in 2017, about 96% of the world's fresh milk comes from cows and buffaloes, while the remaining 4.0% is produced by goats, sheep and camels (FAOStat, 2017).

Noteworthy, milk from all ruminant species contains homologous proteins, which share the same structural, functional, and biological properties [START_REF] Monaci | Milk allergens, their characteristics and their detection in food: a review[END_REF]). Cow's milk contains about 3 g of proteins per 100 mL and includes at least 25 different proteins classified in two main categories according to their solubility at pH 4.6 and 20°C [START_REF] Fox | Milk proteins as food ingredients[END_REF]; the insoluble protein fraction that precipitates are caseins (αS1-casein, αS2-casein, β-casein, κ-casein), while the soluble fraction constitutes the serum characterized by whey proteins (β-lactoglobulin, α-lactalbumin, bovine lactoferrin, bovine serum albumin, and immunoglobulins), corresponding to 80% and 20%, respectively. All the caseins present genetic polymorphisms, which account for the high heterogeneity of such proteins with several identified variants. The latter are characterized by single amino acid substitution, by deletions of peptides fragments and by occurrence of post-translational modifications, such as glycosylation, phosphorylation, or partial hydrolysis, which may affect their specific allergenicity [START_REF] Fox | Milk proteins as food ingredients[END_REF][START_REF] Wal | Structure and function of milk allergens[END_REF] and peptide utility in LC-MS assays. The main allergenic proteins of whey are the globular proteins β-lactoglobulin and α-lactalbumin, which are not phosphorylated and contain intramolecular disulfide bonds that stabilize their structure.

Besides the direct consumption of raw milk, a wide range of derived food products can be manufactured from either raw milk or its condensed forms, such as cheese, butter products, cream products, powdered formulations; in addition, casein and whey protein fractions can also be used independently as ingredients in several commodities, including cheese, bakery products and glues. Therefore, the design of robust methods of analysis tailored to detect potential milk contaminations requires the selection of specific markers tracking independently the two main milk fractions, caseins and whey.

The critical evaluation of data displayed in Fig. 2, pointed out that milk has not been only the most investigated source of food allergens, but also the allergenic food where the highest consensus was achieved in the selection of signature peptides. The milk proteotypic peptides FFV, YLG and HQG (see Table S2 for peptides full sequences) belong to the αS1-casein (Bos d 9), which is considered the most abundant protein in milk. They have been validated in many independent studies for targeted analysis, including several kind of matrices, such as liquid matrices (wine, ice cream), bakery products (bread, cookie, muffin), high fat and polyphenol matrices (chocolate), acidic formulation (tomato sauce), high protein matrices (meat products) etc. In addition, a few signature peptides were also reported for β-casein (Bos d 11), αS2-casein (Bos d 10), and κ-casein (Bos d 12), in the perspective to monitor two different allergenic proteins tracking for the same ingredient. As for whey proteins, main reliable signature peptides belonged to the β-lactoglobulin (β-LG, MW=18.3 kDa), which is the most abundant protein in whey. This protein belongs to the lipocalin superfamily and is one of the best characterized lipid-binding proteins. It possesses three disulfide bridges and is present in two main variants with punctual mutations. β-LG occurs naturally as a mixture of monomers and dimers, but the proportion of monomers increases after heating to 70°C [START_REF] Monaci | Milk allergens, their characteristics and their detection in food: a review[END_REF]. TPE, LVL and LSF were highlighted as the most common signature peptides, already validated into incurred matrices. Noteworthy, the LSF peptide corresponding to the protein C-terminal, despite featuring a cysteine residue, has been selected for its robustness. In addition, some peptide markers were proposed also for α-lactalbumin (VGINYWLAHK, FLDDDLTDDIMCVK, DDQNPHSSNICNISCDK, LDQWLCEK, CEVFR), but none of them was validated in either chocolate-based matrix or thermally processed incurred matrices; therefore, they were excluded from the list of candidate markers at the first selection stage. The specificity of all reported milk peptide markers was confirmed, and some of the sequences showed conservation among proteins from other farmed species, still with no specificity issues with other allergenic food ingredients.

Eggs are, together with milk the most common allergenic foods affecting European children.

Hen's eggs are very common in the human diet as inexpensive source of high quality proteins and they represent a key ingredient in many food products, given its nutritional value and unique functional properties, such as emulsifying, foaming and gelling. Several egg-based formulations have been involved in food manufacturing, such as whole egg, egg white, lysozyme isolate, egg yolk, all containing a different degree of allergenic potential [START_REF] Benedé | Egg proteins as allergens and the effects of the food matrix and processing[END_REF]. The main egg white allergenic proteins are ovomucoid (Gal d 1), ovalbumin (Gal d 2), lysozyme (Gal d 4) and ovotransferrin (Gal d 3), all listed in Table 1, with specific mention to the known posttranslational modifications. Yolk, globally, it is significantly less allergenic than albumen, containing two main allergenic proteins, namely Gal d 5 and Gal d 6, both belonging to the livetins fraction. The latter accounts for about 30% of the yolk proteins, however, relative abundance of Gal d 5 and Gal d 6, is hard to define. Gal d 5 is the main α-livetin, whereas Gal d 6 (protein YPG 42) derives from major β-livetins as C-terminal fragment of vitellogenin 1.

Ovomucoid is one of the most important allergen in the egg white, however it was rarely selected as reporter for egg contamination, in MS based methods. Indeed we retrieved information about a single peptide marker (AFNPVCGTDGVTYDNECLLCAHK, [START_REF] Montowska | Detection of peptide markers of soy, milk and egg white allergenic proteins in poultry products by LC-Q-TOF-MS/MS[END_REF] that was never validated in either chocolate-based matrix or thermally processed incurred matrices, thus excluded from the list of candidate markers at the first stage of the literature review. Ovalbumin, as most abundant protein in egg white, is the most common protein used as marker forhen's egg contamination. It is a phosphoglycoprotein with a molecular mass of 45 kDa belonging to the serpin superfamily, with an intra-molecular disulfide bond between C residues in positions 74 and 121. Noteworthy, besides the most cited peptide GGL, which was monitored in several independent investigations, eight of them including hardto analyze matrices, all the other peptides LTE, ELI and YPI contain amino acids prone to modifications, methionine, asparagine (NG motif) and cysteine, respectively (Fig. 2). In particular, the peptide LTE was alternatively detected either in its native or in the M-oxidized forms depending on the matrix (De [START_REF] De Angelis | Coupling SPE on-line pre-enrichment with HPLC and MS/MS for the sensitive detection of multiple allergens in wine[END_REF]Pilolli et al., 2017a;[START_REF] Pilolli | Development of a mass spectrometry immunoassay for unambiguous detection of egg allergen traces in wines[END_REF][START_REF] Pilolli | In house validation of a high resolution mass spectrometry Orbitrap-based method for multiple allergen detection in a processed model food[END_REF]. The peptide ELI contains the dipeptide NG prone to spontaneous asparagine deamidation. The peptide YPI contains the cysteine residue in position 121 involved in the disulfide bridge. Alternative ovalbumin signature peptides have been also reported, such as HIA and ISQ all displayed in Fig. 2.

Similarly to milk fractions, different egg-derived formulations used in food manufacturing require specific markers to be selected for each part of the allergenic ingredient that could eventually contaminate food commodities. Thus, in addition to egg white markers, it is important to select specific markers of egg lysozyme, industrially applied as protein isolate for its antimicrobial properties, and egg yolk. Only a limited percentage of the collected studies considered this point. As for direct lysozyme detection, we disclosed three main markers, FES, NTD and GTD, which have been validated at least once into incurred baked products, such as cookies and muffins. The egg yolk has been rarely investigated so far, indeed only six studies were collected detecting specific egg yolk proteins, four of them developing a targeted approach [START_REF] Planque | Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs[END_REF]Planque et al., 2017b;[START_REF] New | Simultaneous Analysis of Multiple Allergens in Food Products by LC-MS/MS[END_REF][START_REF] Planque | Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory[END_REF], and two presenting only the identification of potential markers by HR-MS discovery analysis [START_REF] Lee | Determination of Allergenic Egg Proteins in Food by Protein-, Mass Spectrometry-, and DNA-Based Methods[END_REF][START_REF] Gavage | Selection of egg peptide biomarkers in processed food products by high resolution mass spectrometry[END_REF]. Luckily, most of them already tested a wide diversity of food matrices, with different complexity degree, such as cookie, bread, chocolate, salad dressing, spices, ham (see Table S2 for further details), also taking into consideration the ingredient inclusion before food processing. Noteworthy, [START_REF] Gavage | Selection of egg peptide biomarkers in processed food products by high resolution mass spectrometry[END_REF] proposed a systematic investigation of potential egg markers (selected for both egg white and egg yolk), in raw and processed eggbased commodities, promoting the use of a dual enzyme digestion protocol, which benefits from the use of the lysyl endopeptidase in addition to trypsin to provide the same site-specific cleavage with higher proteolytic yield. Focusing on the four targeted investigations [START_REF] Planque | Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs[END_REF]Planque et al., 2017b;[START_REF] New | Simultaneous Analysis of Multiple Allergens in Food Products by LC-MS/MS[END_REF][START_REF] Planque | Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory[END_REF], we disclosed that none of the reported signature peptides traced directly the allergenic protein YGP42, which is the carboxy-terminal portion (284 AAs) cleaved from the primary translation product of vitellogenin-1. Indeed, the peptides YLL and ALL both belong to the amine terminal chain of vitellogenin-1, known as lipovitellin-1. Three additional peptides included in the allergenic protein YGP42 were proposed by [START_REF] Gavage | Selection of egg peptide biomarkers in processed food products by high resolution mass spectrometry[END_REF], TVI, NVN, ATA, by mean of the dual enzyme digestion protocol, which could be considered for further assessment in the development of targeted investigations.

The sequence specificity control by BLAST proved that none of the reported peptide markers was common to other allergenic ingredients, however some of them (especially lysozyme and vitellogenin-2 peptides) presented conserved sequence in proteins from bacteria and from other animal species, unlikely to occur in food commodities.

Further non-allergenic proteins were also proposed to trace for egg yolk contamination, such as the vitellogenin-2, the apolipoprotein B and the apovitellenin-1.

Proteins and peptide markers for legumes: peanut and soybean.

Peanut and soybean are both members of the Fabaceae or Leguminosae family. Legumes represent 27% of the primary crop production worldwide, highly diffused in the human diet as excellent sources of proteins, water soluble fibers, numerous micronutrients, and phytochemicals [START_REF] Smýkal | Legume Crops Phylogeny and Genetic Diversity for Science and Breeding[END_REF]. Peanut is the best characterized legume in regard to its allergenic content, having sixteen proteins, mainly seed storage proteins, classified as allergens with specific isoforms identified and listed in Table 1. As for soybean, the number of characterized allergens is relatively limited, with eight allergens officially registered by the WHO/IUIS Allergen Nomenclature Subcommittee (Allergen Nomenclature, accessed in September 2018). Legume allergens can be grouped into a restricted number of families and superfamilies, and the most relevant allergens are seed storage proteins belonging to the cupin and prolamin superfamilies. The cupin superfamily share a conserved β-barrel structural core domain, which can be single or double; the seed storage proteins 7S and 11S globulins are both bicupins. The prolamin superfamily includes proteins with conserved cysteine residues at specific positions, sharing a common tridimensional structure with four α-helices stabilized by disulfide bonds. This superfamily includes the 2S albumins and non-specific lipid transfer proteins (ns-LTPs). In addition, minor allergens belong to the profilins and Bet v (Betula verrucosa) 1-like superfamilies, which are associated with pollen allergy, the defensins, involved in plant defense against pathogens, and the oleosins, structural proteins of intracellular lipid storage organelles.

Current methods for MS-based peanut detection mainly target Ara h 1 (7S-globulin, vicilin-type), Ara h 2 (2S albumin, conglutin) and Ara h 3 (11S globulin, glycinin) proteins. All peptides reported for these three proteins are highly specific, being conserved only among specific peanut related taxonomies (Arachis ipaensis (130454), Arachis hypogaea (3818), Arachis duranensis (130453)). Ara h 1 is a glycoprotein that makes up 12-16% of the total protein content and forms stable trimers held by non-covalent interactions [START_REF] Palladino | Peanut allergens[END_REF]. Two isoforms has been identified, encoded by two different clones 41 B (Ara h 1.0101) and P17 (Ara h 1 clone P 17), with a sequence identity higher than 90%. Both proteins have an N-terminal 25 amino acid residue signal peptide and a single glycosylation site. The signal peptide drives the nascent protein to the storage vacuole where both the signal itself and a specific portion of the N-terminal is cleaved-off to yield the mature Ara h 1 found in peanut [START_REF] Palladino | Peanut allergens[END_REF]. All the Ara h 1 marker peptides reported so far are shared between the two isoforms, and none of them include the glycosylation site (asparagine residue at position 521 of clone 41B and position 516 of clone P17, see Fig. S1 for details). DLA, VLL and GTG are the most frequently selected, already tested in several incurred and hard-to analyze matrices.

Ara h 2 represents the 5.9-9.2% of the total protein content and can be expressed from different genes into two isoforms Ara h 2.01, and Ara h 2.02, the latter differing for the insertion of a 12 amino acid motif at position 75 in comparison to the Ara h 2.01. In addition, the Allergome database further distinguishes the two isoforms in four variants (Ara h 2.0101, Ara h 2.0102, Ara h 2.0201, Ara h 2.0202), which, besides the signal sequence, differ for a single amino acid mismatch (E vs D) close to the C-terminal portion of the full sequence (position 163 in the Ara h 2.0201 sequence). Furthermore, Ara h 2 undergoes proteolytic processing by peanut proteases resulting in the removal of the C-terminal dipeptide RY. Consequently, the Ara h 2 can be found as a mixture of all the four variants as well as their slightly truncated forms. Four intra-molecular disulfide bonds hold the tridimensional structure of the Ara h 2 and three proline residues are present in their hydroxylated form (see Fig. S2). Interestingly, none of the markers reported in Fig. 3 for Ara h 2 contain the 4-hydroxylproline residues, however all of them contain cysteine residues directly involved in the formation of the four disulfide bridges. In addition, the CMC also contained two methionine residues, susceptible to oxidation. The first three peptides, namely CCN, NLP and CMC are shared among all the isoforms (see Fig. S2), whereas the peptide CDL includes the position affected by single amino acid substitution, thus both the variants CDLEVESGGR (reported peptide) and CDLDVESGGR (potential alternative sequence) can be found in nature with unpredictable proportions. Ara h 3 is a glycinin-like protein (11S) with a molecular mass of 60 kDa for the monomer and occurs in peanuts as hexamer of 360 kDa [START_REF] Palladino | Peanut allergens[END_REF]. The monomer is post-translationally cleaved in 43 kDa acidic and 28 kDa basic subunits, covalently linked by a single disulphide bond. Ara h 3 and Ara h 4, initially considered as different allergenic proteins, have been identified as variants of the same gene, thus they were renamed as isoforms Ara h 3.01 and Ara h 3.02. These are the only two isoforms officially listed by the WHO/IUIS Allergen Nomenclature Subcommittee, whereas further Uniprot entries are classified as Ara h 3 in the Allergome database. A genomic clone encoding Ara h 3 (AF10854) [START_REF] Viquez | Genomic organization of peanut allergen gene, Ara h 3[END_REF] was identified as having four exons. The deduced protein (538 AA in length) showed 93% and 91% identity with the Ara h 3 (isoform Ara h 3.01) and Ara h 4 (isoform Ara h 3.02) [START_REF] Palladino | Peanut allergens[END_REF], respectively. Furthermore, an additional isoform named iso-Ara h 3 was reported as sharing only 70-85% of identity with previously cited sequences (Boldt et al., 2005).

Several peptide markers tracing for Ara h 3 protein were reported so far (see Fig. 3), however, surprisingly for this particular case, the criterion of sequence sharing among known isoforms has not been valued properly in the current literature (see Fig. S3). Indeed, some of proposed markers were not conserved among protein variants, and according to common sense, they should have been excluded, for their limited representativeness. For example, the peptide SPD, which is cited in 13 investigations, is encrypted only in the Ara h 3.01 isoform (and in the genomic clone). Quite surprising was also the selection of the peptide TANELNLLILR in two very recent papers (Planque et al., 2017a;Planque et al., 2017b), which was not encoded by the officially recognized isoforms Ara h 3.01 and Ara h 3.02, but only by the Ara h 3 genomic clone. Noteworthy, the peptide LNA is the only marker fully conserved across the isoforms while, the peptides RPF, QQP are common only to the two main isoforms Ara h 3.01 and Ara h 3.02.

As previously stated, soybean is another edible legume belonging to the Fabaceae family, widely consumed worldwide for its high protein content (approximately 38-40 %). It finds also wide applicability as ingredient in meat/poultry products, bakery and pastry products, dairy products, and edible spreads, as well as additive for a variety of pharmaceutical and industrial applications [START_REF] Verma | A Comprehensive Review of Legume Allergy[END_REF]. As such, the number of occurring soy based formulations is very high, ranging from raw to highly processed and/or hydrolyzed commodities and this makes the absolute quantification of soy contamination in complex matrices quite challenging.

The WHO/IUIS recognized eight soybean proteins as official allergens, including hydrophobic protein (Gly m 1), defensin (Gly m 2), soy profilin (Gly m 3), pathogenesis-related protein (Gly m 4), β-conglycinin (Gly m 5, vicilin, 7S globulin), glycinin (Gly m 6, legumin, 11S globulin), seed biotinylated proteins (Gly m 7) and the 2S albumin protein group (Gly m 8). The major storage proteins, β-conglycinin and glycinin, both belonging to the cupin superfamily, represent alone the 70-80% of the total seed globulin fraction (De Angelis et al., 2017a). β-Conglycinin is 7S globulin glycoprotein containing 5% of carbohydrate moieties and occur as trimers with molecular masses of approximately 180 kDa. Therefore, the Gly m 5 allergen name refers to the complex of three β-conglycinin subunits that were not characterized at the time the name was assigned [START_REF] Pomés | WHO/IUIS Allergen Nomenclature: Providing a common language[END_REF]. The three subunits were first described by [START_REF] Holzhauser | Soybean (Glycine max) allergy in Europe: gly m 5 (beta-conglycinin) and Gly m 6 (glycinin) are potential diagnostic markers for severe allergic reactions to soy[END_REF] labeled as α (67 kDa), α' (71 kDa) and β (50 kDa), and were assessed to combine at different ratios to form the multimeric complexes. The α and α′ subunits are approximately 82% identical, whereas the β-subunit is only 76% identical to the two α subunits. The individual subunits were named as isoallergens Gly m 5.01, Gly m 5.02 and Gly m 5.03 in 2009, with minor variants (e.g. Gly m 5.0301 and Gly m 5.0302 presenting two amino acid mismatches L/F at positions 16 and 198). Interestingly, the in-silico simulation of tryptic digestion showed that the isoallergens Gly m 5.01, Gly m 5.02 and Gly m 5.03 do not share any tryptic peptide with minimum length of 7 AA, which could be selected as a common marker for the βconglycinin subunits. This issue makes the absolute quantification of soybean allergen by synthetic peptide analysis quite challenging since the required conversion factors should base on the knowledge of the specific combination of α, α′ and β-subunits occurring in the sample under investigation. As expected by in-silico prediction, none of the reported peptide markers for Gly m 5 displayed in Fig. 3 were conserved among the three isoallergens (see Fig. S4).

Interestingly, an alternative solution, in this case, would be to select unique markers for each subunit in order to disclosethe specific combination of α, α′ and β-monomers within the trimeric complex. In this frame, the peptide LIT would uniquely trace for Gly m 5.01, the peptides QQQ and DSYNLQ would uniquely trace for Gly m 5.02, and the peptide DSYNLH would uniquely trace for Gly m 5.03 (noteworthy, the latter is conserved between the two variants Gly m 5.0301 and Gly m 5.0302). Attention should be paid to the peptide AIV, even if theoretically unique for Gly m 5.01, since an homologous peptide sequence is expressed in both variants of Gly m 5.03, with a single amino acid substitution L/I at position P19, which could not be discriminated by SRM acquisition. None of the aforementioned peptides contain amino acid residues with naturalpost-translational modification.

In most soybean varieties, the glycinin accounts for over 50% of seed storage proteins representing the predominant protein fraction (De Angelis et al., 2017a). Glycinin has a complex hexameric structure with a molecular weight ranging between 320 to 360 kDa. At least five genes encode the monomeric subunits [START_REF] Nielsen | Characterization of the glycinin gene family in soybean[END_REF] and the Gly m 6 allergen can be an arrangement of these five proteins, which have individual IgE binding properties [START_REF] Holzhauser | Soybean (Glycine max) allergy in Europe: gly m 5 (beta-conglycinin) and Gly m 6 (glycinin) are potential diagnostic markers for severe allergic reactions to soy[END_REF]. Each monomer consists of two specific polypeptide chains, one acidic (40 kDa) (A) and one basic (20 kDa) (B), linked together by disulfide bonds. Each monomer can be one of five subunits, namely glycinin G1 (A1aB1b), glycinin G2 (A2B1a), glycinin G3 (A1bB1a), glycinin G4 (A5A4B3) and glycinin (A3B4) [START_REF] Wang | Advances of Research on Glycinin and β-Conglycinin: A Review of Two Major Soybean Allergenic Proteins[END_REF], being renamed as five isoallergens Gly m 6.0101, Gly m 6.0201, Gly m 6.0301, Gly m 6.0401 and Gly m 6.0501, respectively, in 2009.

Similarly to what was already discussed for the quantification of Gly m 5 allergen, the unpredictable composition of the Gly m 6 hexameric complex makes general conversion factors a hard task to achieve. The peptide marker selection carried out so far in current literature completely neglected the different sequence expression in known isoallergens. Indeed, the most frequently used markers, VFD, VLI, and EAF, are expressed uniquely in Gly m 6.0101, whereas the peptide SQS, cited in 8 independent investigations, is conserved across the three isoforms Gly m 6.0101, Gly m 6.0201, Gly m 6.0301, and the peptide IST (cited in 7 independent investigations) is common to the isoforms Gly m 6.0401 and Gly m 6.0501 (see Fig. S5).

Proteins and peptide markers for tree nuts: hazelnut and almond

Hazelnut and almond allergens were selected in this project as most relevant members of the tree nut category according to the European legislation. Similar to peanut and soybean, the main proteins involved in tree nut allergy belong to cupin (legumins-11S globulin and vicilin-7S globulin) and prolamin (conglutin-2S albumin and ns-LTPs) superfamilies. Several hazelnut allergens have been identified and are already partly applied in component resolved diagnosis [START_REF] Geiselhart | Tree nut allergens[END_REF]. However, only partial information have been collected so far about the occurrence of potential allergen isoforms and their complete sequencing, even if their existence is likely, given the high genetic diversity of plant organisms. Even the limited information available for almond allergens, the provided following discussion about the peptide marker selection was driven to the best of the current knowledge for these two allergens, and relevant homologous plant proteins.

WHO/IUIS database included eight hazelnut allergens all listed in Table 1. According to the EuroPrevall population-based survey, including allergic subjects from twelve European cities, the predominance of specific hazelnut allergens is correlated to the geographical area [START_REF] Pastorello | Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results[END_REF], as well as to the age of the sensitized individuals [START_REF] Verweij | Age-dependent sensitization to the 7Svicilin like protein Cor a 11 from hazelnut (Corylus avellana) in a birch-endemic region[END_REF].

In the Mediterranean areas, hazelnut allergy is mainly linked to Cor a 8, Cor a 14, Cor a 9, Cor a 11 [START_REF] Schocker | Recombinant lipid transfer protein Cor a 8 from hazelnut: a new tool for in vitro diagnosis of potentially severe hazelnut allergy[END_REF]Blanc et al., 2015). Cor a 8 is a nsLTP type 1, as mature protein, consisting of a unique polypeptide chain of 92 amino acid residues with eight strictly conserved cysteines forming four intramolecular disulfide bridges. Cor a 14 (2S albumin) is a heterodimeric protein with characteristic structural features, including four α-helices and eight cysteine residues engaged in four disulfide bridges [START_REF] Pfeifer | Cor a 14, the allergenic 2S albumin from hazelnut, is highly thermostable and resistant to gastrointestinal digestion[END_REF]. The precursor protein is posttranslationally cleaved into a large and a small subunits linked by disulfide bonds. The Nterminal glutamine shows the cyclization to pyroglutamic acid and the small subunit is variably truncated at the C-terminal, leading to a high micro-heterogeneity [START_REF] Pfeifer | Cor a 14, the allergenic 2S albumin from hazelnut, is highly thermostable and resistant to gastrointestinal digestion[END_REF]. Cor a 9, as 11S legumin, exhibits some homology with other tree nut allergens, such as cashew Ana o 2, peanut Ara h 3 and the soybean glycinin (Gly m 6). The monomer consists of two polypeptide chains (acidic and basic chains), cleaved post-translationally by site specific endopeptidases, and linked together by a single intermolecular disulfide bond. The WHO/IUIS recorded a single isoform of the Cor a 9 protein corresponding to the Uniprot accession Q8W1C2 and labelled as Cor a 9.0101, first identified in 2002 as IgE-binding protein by 2D-Western blotting, using sera of hazelnut allergic patients [START_REF] Beyer | Identification of an 11S globulin as a major hazelnut food allergen in hazelnut induced systemic reaction[END_REF]. In 2013, a new isoform of the same protein, with a 514 AA full-length sequence, was registered (Uniprot accession A0A0A0P7E3) sharing 96.5% homology [START_REF] Grishina | Isoform of a Major hazelnut food allergen Cor a 9 (11S Globulin)[END_REF]. This isoform has been added to the Allergome database with the general label of Cor a 9. Additionally, at least another 55 kDa protein, with high IgE-reactivity only in the alkaline chain (20.7 kDa), has been identified by [START_REF] Nitride | Proteomic and immunological characterization of a new food allergen from hazelnut (Corylus avellana)[END_REF]. In this investigation, shotgun proteomics allowed de novo sequencing of six peptides of the basic chain and their assignment to 11S protein isoform sharing high homology with 11S globulin-like proteins from several plant organisms, including the canonical hazelnut one. Starting from this background, we reviewed the current literature tracing the Cor a 9 selected markers. The peptide WLQ should be excluded by any further discussion since conserved sequence among different tree nut species (hazelnut, pistachio, pecan, and walnut). Besides this, we assessed that all the proposed signature peptides shared 100% identity between the official Cor a 9.0101 (Q8W1C2) sequence and its registered isoform A0A0A0P7E3 (see Fig. S6). However, considering the further potential isoform only partially sequenced by [START_REF] Nitride | Proteomic and immunological characterization of a new food allergen from hazelnut (Corylus avellana)[END_REF], three out of the seven reported peptides might be excluded, as non-conserved regions and, noteworthy, this exclusion would involve two of the most cited peptides INT and ALP. Therefore, a wider characterization of most common isoform occurring by natural genetic diversity is urgently needed. In addition, the peptide VQV contains the NG motif, which makes the asparagine residues highly susceptible to deamidation.

Cor a 11 is a 7S globulin like protein, featuring a trimeric structure of MW about 150/190kDa with subunits of about 50kDa. The WHO/IUIS lists a single isoform labelled as Cor a 11.0101, corresponding to the Uniprot entry Q8S4P9, which includes a single glycosylation site occurring at the asparagine residue. Three peptide markers have been reported for this protein, namely LLS, AFS, and ELA, and none of them includes amino acid residues prone to modifications.

As for almond, six identified almond allergens, Pru du 3 (ns-LTP), Pru du 4 (profilin), Pru du 5 (60 S ribosomal protein), and Pru du 6 (legumin), are included in the WHO-IUIS allergen list.

The specificity in the analytical detection of this allergen is particularly challenging due to both a limited availability of full-length sequences in official database and due to the high homology of almond proteins (taxonomy Prunus dulcis) within the Prunus taxonomy genus, mainly Prunus persica (peach) [START_REF] Inman | Development of a LC-MS method for the discrimination between trace level Prunus contaminants of spices[END_REF]. For example, the almond allergen Pru du 3.0101 displays 99% amino acid sequence identity to a nsLTP from peach (UniProtKB M5W0S9, not listed in the IUIS) and 57% to two other peach nsLTP isoforms, Pru p 3.0101 and Pru p 3. 0102. Two native isoforms have been identified for the almond profilin (Pru du 4), but its relevance is mainly restricted to pollen-associated almond allergy. Pru du 5 has been identified as an 11.4 kDa 60S ribosomal protein involved in protein biosynthesis [START_REF] Abolhassani | cDNA cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (Prunus dulcis)[END_REF]; nevertheless, the clinical relevance of this allergen is unknown and requires further research. Finally, Pru du 6, a legumin-like protein, also called amandin, is the first and best biophysically characterized allergen in almond to date [START_REF] Geiselhart | Tree nut allergens[END_REF]. Two cDNA clones encoding almond legumins named prunin-1 (61.0 kDa) and prunin-2 (55.9 kDa) have been isolated, both included in the WHO/IUIS database as Pru du 6.0101 and Pru du 6.0201. By in-silico simulation of the enzymatic digestion with trypsin specific cleavage, it was assessed that no tryptic peptide with minimum length of 7 AA presents full identity between the two isoforms Pru du 6.0101 and Pru du 6.0201 (see Fig. S7). In addition, Allergome database also includes two variants of Pru du 6.0101 and Pru du 6.0201, registered with the Uniprot entries Q43607 and Q43608, respectively, which share 99% identity with the relevant official allergen isoform, featuring only few amino acid substitutions (see Fig. S7). In Fig. 3, we presented the signature peptides reported so far for almond detection by SRM analysis all belonging to the Pru du 6 protein, either to the Pru du 6.0101 or to the Pru du 6.0201 isoallergens. Noteworthy, most of the selected peptides highlighted with asterisks presented specificity issues, since the BLAST search against non-redundant protein database showed 100% sequence identity with homologous proteins from Prunus persica, Prunus mume or Prunus avium, (see Fig. S8 andS9). In light of this, we kept in the list reported in Fig. 3 also the two markers QQG both VQV, even if they were not validated in incurred or chocolate based matrices, as only candidates, together with TDE, which feature uniqueness for Prunus dulcis species. Interestingly, none of the reported peptides included cysteine residues involved in the disulfide bridges, whereas both the unique peptides VQV and TDE contain the motif NG that make them susceptible to spontaneous deamidation. In addition, the sequences VQVVNENGDPILDDEVR and ALPDEVLQNAFR, belonging to the Pru du 6.0201 isoform were not conserved in the alternative allergen variant registered as Q43608 in Uniprot database, both presenting single amino acid substitutions (D/N and N/T, respectively) and the peptide variant VQVVNENGDPILNDEVR is shared with Prunus persica (see sequence alignment in Fig. S9).

List of candidate markers agreed by ThRAll partners

The thorough discussion presented in the previous section allowed the selection of a restricted list of candidate signature peptides that can be fruitfully employed for allergen detection in highly complex and processed foods with a high-confidence on identification accuracy. This list representing the consensus table of the consortium is reported in Table 2 and will be further validated for its reliability in the model matrices selected within the ThRAll project by discovery experiments by untargeted HR-MS/MS analysis. As already mentioned, the preliminary marker selection here performed by means of the critical evaluation of current literature represents only the first step of the dual approach devised for the ThRAll project. The experimental validation of the candidate markers by MS/MS analysis is a mandatory step in method development because several empirical features, such as matrix composition, extraction/digestion conditions, and instrumental parameters might affect both the specificity and sensitivity of the final analytical method. Interferences in the chromatographic analysis from complex food background and information about processing effects on the ingredient itself or finished food products should be traced to avoid false-negative and false-positives. Such information cannot be disclosed merely by any in-silico based approach.

The Table 2 was populated with all the information about sequence sharing among isoforms/variants per each discussed peptide.

As for the protein homology check among different taxonomies carried out on plant-derived allergens, we concluded that according to the current knowledge of full-length protein sequences, none of the signature peptides selected for plant-derived allergens (Table 2) is affected by homology issues. To the best of the current knowledge, the refined list of peptides provided very high detection specificity. For sake of clarity in Table S3, we reported a short list of peptide markers identified that might experience this kind of interference from other homologous proteins, disclosing both the targeted and the interfering precursor ions, as well as the shared transitions.

As for Pru du 6 peptides, with high sequence identity with homologous proteins from Prunus persica, Prunus mume and Prunus avium, only two peptide markers were kept, including the unique peptide TDE, even if encrypting the NG motif, since it can still be useful for confirmative purposes.

Finally, in Table 2 we also collected information about the reported precursor ions and the transitions monitored. Both the preferential ionization state of the target peptide and its typical fragmentation pattern depend not only on the specific amino acid sequence, but also on instrumental parameters such as the ionization source, the MS platform, and the activation mode; therefore, after proper marker selection, the ionization and fragmentation features need to be assessed on the specific MS platform involved in the study. From a general point of view, we highlighted that most of the selected signature peptides were detected as double charged ions, which indeed are very stable and reproducible both in their ionization yield and in the fragmentation pattern, whereas an heterogeneous scenario was disclosed as for transitions selection. Either two or three transitions per peptide were acquired, in some cases the two monitored fragments are simply two different ionization states of the same transition (yn + and yn ++ ). This behavior is typical of proline containing peptides that upon collisional induced fragmentation preferentially break the peptide bond at the N-terminal proline site, releasing the same transition as both single and double charged ions [START_REF] Ma | De novo sequencing and homology searching[END_REF]. This transition is usually very sensitive, but the ratio (yn + /yn ++ ) between the two ions is not fixed, and may change upon different instrumental conditions. Additionally, retrieving the SRM instrumental details for the reported papers we also disclosed that in some investigations quite small fragments have been selected, namely y1 + , b2 + , a2 + , notwithstanding their low selectivity and susceptibility to interference from the background.

In light of the harmonization of detection method, and given the heterogeneous picture retrieved for instrumental set up, we will select at least three transitions for each marker, preferably belonging to the yn and bn series. Indeed, y-and b-ion series together would allow read out of the full peptide sequence from the MS/MS spectrum in the two directions starting from the Cand the N-terminus, respectively [START_REF] Ma | De novo sequencing and homology searching[END_REF]; in addition, the two series together would account for most of the MS/MS spectrum ion intensity in both HCD and CID fragmentations (Michalski et al., 2012), with very high stability of the y-type transitions.

Noteworthy, the monitored fragments should actually correspond to different transitions with a minimum fragment length of 3 amino acids, given the minimum length for peptide markers of 7 amino acids, in order to guarantee the maximum specificity and sequence coverage to the final developed method.

Future development of this work, will be the experimental validation of the reported preliminary list of candidate markers. In particular, discovery high resolution MS/MS analysis will be carried out directly on the two matrices, namely chocolate bar and broth powder as model hard-to analyze food matrices, produced in a food pilot plant and incurred at the high contamination level. The experimental list of identified peptides belonging to the allergenic ingredients will be compared with the literature based selection either confirming or proposing new options for the SRM set up.

Conclusions

In this work we described the results of our comprehensive evaluation of the existing literature on food allergen detection by MS based methods, concerning the selection of peptide markers.

We collected all information about the six allergens selected for the ThRAll project, namely cow's milk, hen's egg, peanut, soybean, hazelnut and almond. The selected peptides were critically discussed according to specific considerations and recommended criteria for marker selection that we promote in perspective harmonization of analytical methods development.

The in-depth analysis of current knowledge allowed the selection of a restricted list of candidate signature peptides, already validated in several independent analyses, which could be monitored with high confidence. This preliminary list will be validated in the two model matrices selected within the ThRAll project, in a separate investigation for its final confirmation by specific discovery experiments with untargeted HR-MS/MS analysis.

FIGURE AND TABLE CAPTIONS

Figure 1. Analytical workflow describing step-by-step the literature review and the critical evaluation of the proposed peptide markers in order to compile a final list of highly reliable signature peptides to be tested for the ThRAll project.

FIGURES AND TABLES

Figure 1 (color online version only) 692.9 (+2) 692.9 (+2) 692.9 (+2) 1384.6 (+1) 692.9 (+2) 692.9 (+2) 692.6 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.9 (+2) 692.8 (+2) Heick et al., 2011a[START_REF] Heick | First screening method for the simultaneous detection of seven allergens by liquid chromatography mass spectrometry[END_REF][START_REF] Losito | Development of a Method for the Quantification of Caseinate Traces in Italian Commercial White Wines Based on Liquid Chromatography-Electrospray Ionization-Ion Trap-Mass Spectrometry[END_REF][START_REF] Lutter | Development and Validation of a Method for the Quantification of Milk Proteins in Food Products Based on Liquid Chromatography with Mass Spectrometric Detection[END_REF][START_REF] Monaci | Multi-allergen quantification of fining-related egg and milk proteins in white wines by high-resolution mass spectrometry[END_REF][START_REF] Gu | A rapid solid-phase extraction combined with liquid chromatography-tandem mass spectrometry for simultaneous screening of multiple allergens in chocolates[END_REF]Ke et al., 2017 Qi et [START_REF] Lutter | Development and Validation of a Method for the Quantification of Milk Proteins in Food Products Based on Liquid Chromatography with Mass Spectrometric Detection[END_REF][START_REF] Monaci | Multi-allergen detection in food by micro high-performance liquid chromatography coupled to a dual cell linear ion trap mass spectrometry[END_REF][START_REF] Parker | Multi-allergen Quantitation and the Impact of Thermal Treatment in Industry-Processed Baked Goods by ELISA and Liquid Chromatography-Tandem Mass Spectrometry[END_REF][START_REF] Yang | Development of an SI-traceable HPLC-isotope dilution mass spectrometry method to quantify β-lactoglobulin in milk powders[END_REF][START_REF] Ji | Development of a liquid chromatography-tandem mass spectrometry method for simultaneous detection of the main milk allergens[END_REF]Boo et al., 2018Pilolli et al., 2018 New et al., [START_REF] Azarnia | Detection of ovalbumin in egg white, whole egg and incurred pasta using LC-ESI-MS/MS and ELISA[END_REF][START_REF] Mattarozzi | Investigation of different sample pre-treatment routes for liquid chromatography-tandem mass spectrometry detection of caseins and ovalbumin in fortified red wine[END_REF][START_REF] Monaci | Multi-allergen quantification of fining-related egg and milk proteins in white wines by high-resolution mass spectrometry[END_REF][START_REF] Monaci | Multi-allergen detection in food by micro high-performance liquid chromatography coupled to a dual cell linear ion trap mass spectrometry[END_REF]Parker et al., 2015 Pilolli et al., [START_REF] Chassaigne | Proteomics-Based Approach To Detect and Identify Major Allergens in Processed Peanuts by Capillary LC-Q-TOF (MS/MS)[END_REF][START_REF] Bignardi | Determination of peanut allergens in cereal-chocolate-based snacks: metal-tag inductively coupled plasma mass spectrometry immunoassay versus liquid chromatography/electrospray ionization tandem mass spectrometry[END_REF][START_REF] Parker | Multi-allergen Quantitation and the Impact of Thermal Treatment in Industry-Processed Baked Goods by ELISA and Liquid Chromatography-Tandem Mass Spectrometry[END_REF]Pedreschi et al., Sayers et al., 2016Sayers et al., 2018 Boo et al.,

- y5 + b2 + b2 + - - - - - 423.3 - - - - - - - y3 + - -

Careri

Figure 2 .

 2 Figure 2. Citation overview of the peptides markers used for milk and egg allergens, which

Figure 3 .

 3 Figure 3. Citation overview of the peptides markers used for peanut, soybean, hazelnut and

Table 1 .

 1 Summary of the food allergens proteins recorded for the six allergen under investigations. Acronyms: PTM, post-translational modifications; IUIS, International Union of

	Immunological	Societies.	*information	gathered	from	Uniprot	Database
	(https://www.uniprot.org/, Accessed on September 19 th 2018).

Table 2 .

 2 Preliminary list of signature peptides selected by the Consortium according to the QqQ, triple quadrupole; IT, ion trap; QToF, quadrupole time-of flight; Q-Orbitrap, quadrupole-Orbitrap, LIT, linear ion trap. tandem mass spectrometry multiallergen trace analysis in foods. Journal of Chromatography A, 1217, 7579-7585. https://doi.org/10.1016/j.chroma.2010.10.037.

	analytical workflow presented in Figure 1, which have been monitored in targeted investigations
	with relevant information on allergens isoforms/variants, peptide specificity, and acquisition
	related technical details (MS platform, precursor ion and selected transitions). The presence of
	amino acid residues prone to modification was highlighted with a different font (bold and

underlined). The transition selected as quantifier ion (when specified) was marked with an asterisk. Acronyms: E.,

Hoffmann-Sommergruber, K., Vieths, S., Rigby, N., Mills, E. N. C., & Adel-Patient, K. (2015)

. Further studies on the biological activity of hazelnut allergens. Clinical and Translational Allergy, 5, 26. https://doi.org/10.1186/s13601-015-0066-7. Boldt, A., Fortunato, D., Conti, A., Petersen, A., Ballmer-Weber, B., Lepp, U., Reese, G., & Becker, W. M. (2005). Analysis of the composition of an immunoglobulin E reactive high molecular weight protein complex of peanut extract containing Ara h 1 and Ara h 3/4. Proteomics, 5, 675-686. https://doi.org/10.1002/pmic.200401150. Boo, C. C., Parker, C. H., & Jackson, L. S. (2018). A Targeted LC-MS/MS Method for the Simultaneous Detection and Quantitation of Egg, Milk, and Peanut Allergens in Sugar Cookies.

Journal of AOAC International, 101, 108-117. https://doi.org/10.5740/jaoacint.17-0400. Bucchini, L.,

Guzzon, A., Poms, R., Senyuva, H. (2016)

. Analysis and critical comparison of food allergen recalls from the European Union, USA, Canada, Hong Kong, Australia and New

Table 1

 1

	Allergenic	Protein	Allergen Name	MW (kDa)	Number of known PTMs*	Isoforms and	IUIS	GenBank Protein	UniProt
	ingredient					Variants	(YES/NO)
	Milk	αS1-Casein	Bos d 9	23.6	n° 9 Ser	Bos d 9.0101	Yes	NP_851372	P02662
					phosphorylationsphosphoryl
					ations
		αS2-Casein	Bos d 10	25.2	n° 13 Ser	Bos d 10.0101	Yes	NP_776953	P02663
					phosphorylationsphosphoryl
					ations
		β-Casein	Bos d 11	24	n° 5 Ser	Bos d 11.0101	Yes	XP_005902099	P02666
					phosphorylationsphosphoryl
					ations
		κ-Casein	Bos d 12	19	n°1 N-terminal Gln	Bos d 12.0101	Yes	NP_776719	P02668
					cyclization to pyroglutamate
					n° 3 Disulfide Bonds
					n° 3 Ser
					phosphorylationsphosphoryl
					ations
					n° 1 Thr phosphorylation
					n° 6 Thr glycosylations
					n° 2 Ser glycosylations
		α-Lactalbumin	Bos d 4	14.2	n° 4 Disulfide Bonds	Bos d 4.0101	Yes	AAA30615	P00711
					n° 1 Asn glycosylation
		β-Lactoglobulin	Bos d 5	18.3	n° 3 Disulfide Bonds	Bos d 5.0101	Yes	CAA32835	P02754
		Bovine serum albumin	Bos d 6	66.3	n° 17 Disulfide Bonds	Bos d 6.0101	Yes	AAA51411	P02769
					n° 6 Ser phosphorylations
					n° 4 Thr phosphorylations
					n° 2 N6-succinyllysine
					n° 1 Lys methylation
		Immunoglobulins	Bos d 7	160		Bos d 7.0101	Yes
	Egg	Ovalbumin	Gal d 2	44	Initial Met Removal	Gal d 2.0101	Yes	CAA23682	P01012
					n°1 Gly acetylation
					n° 2 Ser phosphorylations
					n° 1 Disulfide Bond
					n° 1 Asn glycosylation
		Ovomucoid	Gal d 1	28	n° 9 Disulfide Bonds	Gal d 1.0101	Yes	P01005	P01005
					n° 5 Asn glycosylations
		Ovotransferrin	Gal d 3	78	n° 15 Disulfide Bonds	Gal d 3.0101	Yes	CAA26040	P02789
					n° 1 Asn glycosylation
		Lysozyme C	Gal d 4	14	n° 4 Disulfide Bonds	Gal d 4.0101	Yes	CAA23711	P00698
		Serum Albumin	Gal d 5	69	n° 17 Disulfide Bonds	Gal d 5.0101	Yes	CAA43098	P19121
					n° 1 Asn glycosylation
		YGP42	Gal d 6	35	n° 3 Asn glycosylations	Gal d 6.0101	Yes	BAA13973	chain 1628-1912
									of the P87498
		Myosin light chain 1f	Gal d 7	22	n° 1 Ala trimethylation	Gal d 7.0101	Yes	K02608.1, K02609.1	P02604
								and K02610.1
		alpha-parvalbumin	Gal d 8	11.8	NA	Gal d 8.0101	Yes	CAX32963	C1L370
		Beta-enolase	Gal d 9	50	n°1 Ser acetylation	Gal d 9.0101	Yes	NP_990450	P07322

Table 2

 2

	Target	Peptide Sequence	Specificity (Tax ID)	MS platform	Precursor	Transition 1	Transition 2	Transition 3	References
	protein	Isoallergens/variants
	Bos d 9 -	FFVAPFPEVFGK	Bos taurus (9913)	QqQ	693.3 (+2)
	α-S1-Casein	Bos d 9.0101	Bos mutus (72004)	QqQ
			Bubalus bubalis (89462)	QqQ
				IT
				IT
				Orbitrap
				Orbitrap
				Q-ToF
				Q-ToF
				Dual LIT
				QqQ
				QqQ
				Dual LIT / Orbitrap
				Dual LIT
				QqQ
				QqQ
				QqQ
				QqQ
				QqQ
				QqQ
				QqQ
				Q-Orbitrap
				Dual LIT
				QqQ
				QqQ
				QqQ

[START_REF] Korte | MRM3-based LC-MS multi-method for the detection and quantification of nut allergens[END_REF][START_REF] Pilolli | In house validation of a high resolution mass spectrometry Orbitrap-based method for multiple allergen detection in a processed model food[END_REF]

Acknowledgements

This project has received financial support from the European Food Safety Authority (EFSA), Grant GP/EFSA/AFSCO/2017/03 and the UK Food Standard Agency (FSA), Grant FS101206 Development of Quality Control Materials for Food Allergen Analysis The present paper, however, is under the sole responsibility of the authors. The positions and opinions presented in this article are those of the authors alone and do not necessarily represent the views/any official position or scientific works of EFSA. To find out more about EFSA guidance documents and other scientific outputs of EFSA, please consult its website at: http://www.efsa.europa.eu.

The authors are thankful to Dr. Michal Walker (from LGC) for the fruitful exchanges on this topic.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the Authors.

Competing interests 691

The Authors declare they have no competing interest.