**2. Characterization of the peptides deriving from gluten digestion**

Differences in gluten coding genes have been extensively characterized, besides for their tech‐ nological and functional implication in baked products, also for evaluating how much the wheat genetic characteristics can impact on the final immunotoxicity of gluten. One of the most studied immunogenic peptides, the 33‐mer LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, has been demonstrated to be encoded by the 6D chromosome, thus being absent in dip‐ loid and tetraploid *Triticum* species. The lacking of genes encoding for some immunogenic sequences in diploid/tetraploid varieties has also found evidences in T‐cell assays performed on gluten chymotryptic digests that gave different results among the species tested [19]. A lot of efforts have been made in the last years in the direction of decreasing wheat toxicity for celiac patients, for example, making use of *ex vivo* organ cultures and immunohistochemistry assays [20]. The K562 cell agglutinating activity of wheat has been known for many years [21] to screen cereal toxicity for celiac patients [22]. The majority of the studies performed in the past to assess gluten toxicity with immunochemical methods, the main immunotoxic peptides were identified, and in some cases, also a complete characterization of the diges‐ tion mixture was obtained [23]. In the recent years, the advances in the field of mass spec‐ trometry allowed more accurate peptides identification, especially in the case of recombinant gliadin and synthetic peptides [24–27]. Other studies took in consideration purified gliadins, to identify the target peptides of tissue transglutaminase [28]. Peptide mixtures obtained by gluten digests are very complex, since than more than 40 components are present both for gliadins and glutenins [29]. The composition of peptide mixtures derived from simulated gastrointestinal digestion of the whole gliadin fraction of wheat was recently investigated [30]. The gliadin fraction was extracted with 70% ethanol in water and then desiccated. The *in vitro* digestion was performed using the three main proteases of the gastrointestinal tract: pepsin in the gastric phase (3 h) and chymotrypsin/trypsin in the intestinal phase (4 h). All the enzymes were used at their optimal temperature (37°C) and pH (respectively 2 and 7.2), using an enzyme:substrate ratio of 1:100. The determination of the amino acid sequence of the glu‐ ten‐derived peptides was achieved using reverse phase chromatography (High Performance Liquid Chromatography [HPLC], Ultra Performance Liquid Chromatography [UPLC] and Micro‐High Performance Liquid Chromatography [μHPLC]) coupled with different types of mass spectrometer (both high and low resolution, e.g., Linear Trap Quadrupole (LTQ) OrbiTrap, single and triple quadrupoles) to achieve the desired resolving power.

This extensive characterization (**Table 1**) gives useful information for a better understand‐ ing of the peptides that presumably come in contact with the intestinal mucosa, triggering the immunological response in celiac patients. Immunogenic peptides are those containing sequences known in literature to elicit the adaptive immune response, through recognition by the HLA‐DQ2 (or DQ8) of the antigen presenting cells (APC), leading to stimulation of T cell response. As it is shown in **Table 1**, the identified immunogenic peptides derive mainly from α‐gliadin, in particular from the region between the 56th to the 88th amino acid. Toxic pep‐ tides are those containing sequences known in literature to elicit the innate immune response: their interaction with epithelial cells, macrophages and dendritic cells leads to the up regula‐ tion of different cell mediators, the most important one being interleukin‐15. Also in this case, most of the peptides were identified as deriving from α‐gliadin, more specifically from the N‐terminal region. Using the isotopically labelled internal standard method, peptides con‐ taining sequences involved in celiac disease can be quantified: these data can be very helpful for interpretation of the results of immunological assays, since the different response can be due both to different epitopes generation in terms of amino acid sequence and to a different relative amount of pathogenic peptides.

**2. Characterization of the peptides deriving from gluten digestion**

314 Wheat Improvement, Management and Utilization

OrbiTrap, single and triple quadrupoles) to achieve the desired resolving power.

This extensive characterization (**Table 1**) gives useful information for a better understand‐ ing of the peptides that presumably come in contact with the intestinal mucosa, triggering the immunological response in celiac patients. Immunogenic peptides are those containing sequences known in literature to elicit the adaptive immune response, through recognition by the HLA‐DQ2 (or DQ8) of the antigen presenting cells (APC), leading to stimulation of T cell response. As it is shown in **Table 1**, the identified immunogenic peptides derive mainly from α‐gliadin, in particular from the region between the 56th to the 88th amino acid. Toxic pep‐ tides are those containing sequences known in literature to elicit the innate immune response: their interaction with epithelial cells, macrophages and dendritic cells leads to the up regula‐ tion of different cell mediators, the most important one being interleukin‐15. Also in this case,

Differences in gluten coding genes have been extensively characterized, besides for their tech‐ nological and functional implication in baked products, also for evaluating how much the wheat genetic characteristics can impact on the final immunotoxicity of gluten. One of the most studied immunogenic peptides, the 33‐mer LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, has been demonstrated to be encoded by the 6D chromosome, thus being absent in dip‐ loid and tetraploid *Triticum* species. The lacking of genes encoding for some immunogenic sequences in diploid/tetraploid varieties has also found evidences in T‐cell assays performed on gluten chymotryptic digests that gave different results among the species tested [19]. A lot of efforts have been made in the last years in the direction of decreasing wheat toxicity for celiac patients, for example, making use of *ex vivo* organ cultures and immunohistochemistry assays [20]. The K562 cell agglutinating activity of wheat has been known for many years [21] to screen cereal toxicity for celiac patients [22]. The majority of the studies performed in the past to assess gluten toxicity with immunochemical methods, the main immunotoxic peptides were identified, and in some cases, also a complete characterization of the diges‐ tion mixture was obtained [23]. In the recent years, the advances in the field of mass spec‐ trometry allowed more accurate peptides identification, especially in the case of recombinant gliadin and synthetic peptides [24–27]. Other studies took in consideration purified gliadins, to identify the target peptides of tissue transglutaminase [28]. Peptide mixtures obtained by gluten digests are very complex, since than more than 40 components are present both for gliadins and glutenins [29]. The composition of peptide mixtures derived from simulated gastrointestinal digestion of the whole gliadin fraction of wheat was recently investigated [30]. The gliadin fraction was extracted with 70% ethanol in water and then desiccated. The *in vitro* digestion was performed using the three main proteases of the gastrointestinal tract: pepsin in the gastric phase (3 h) and chymotrypsin/trypsin in the intestinal phase (4 h). All the enzymes were used at their optimal temperature (37°C) and pH (respectively 2 and 7.2), using an enzyme:substrate ratio of 1:100. The determination of the amino acid sequence of the glu‐ ten‐derived peptides was achieved using reverse phase chromatography (High Performance Liquid Chromatography [HPLC], Ultra Performance Liquid Chromatography [UPLC] and Micro‐High Performance Liquid Chromatography [μHPLC]) coupled with different types of mass spectrometer (both high and low resolution, e.g., Linear Trap Quadrupole (LTQ)


**Table 1.** Most abundant immunogenic and toxic peptides identified in the digested prolamin extracts (known immunogenic and toxic sequences are underlined), together with an indication of the protein of origin and of their relative abundance in the different types of wheat (durum wheat: *Triticum durum*; common wheat: *Triticum aestivum*).
