Proteomics, Peptidomics, and Immunogenic Potential of Wheat Beer

Article pubs.acs.org/JAFC

Proteomics, Peptidomics, and Immunogenic Potential of Wheat Beer (Weissbier) Gianluca Picariello,*,† Gianfranco Mamone,† Adele Cutignano,‡ Angelo Fontana,‡ Lucia Zurlo,§ Francesco Addeo,† and Pasquale Ferranti†,§ †

Istituto di Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche, Via Roma 64, I-83100 Avellino, Italy Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, I-80078 Pozzuoli (Napoli), Italy § Dipartimento di Agraria, Università di Napoli “Federico II”, Parco Gussone, I-80055 Portici (Napoli), Italy ‡

S Supporting Information *

ABSTRACT: Wheat beer is a traditional light-colored top-fermenting beer brewed with at least 50% malted (e.g., German Weissbier) or unmalted (e.g., Belgian Witbier) wheat (Triticum aestivum) as an adjunct to barley (Hordeum vulgare) malt. For the first time, we explored the proteome of three Weissbier samples, using both 2D electrophoresis (2DE)-based and 2DE-free strategies. Overall, 58 different gene products arising from barley, wheat, and yeast (Saccharomyces spp.) were identified in the protein fraction of a representative Weissbier sample analyzed in detail. Analogous to all-barley-malt beers (BMB), barley and wheat Z-type serpins and nonspecific lipid transfer proteins dominated the proteome of Weissbier. Several α-amylase/trypsin inhibitors also survived the harsh brewing conditions. During brewing, hundreds of peptides are released into beer. By liquid chromatography−electrospray tandem mass spectrometry (LC-ESI MS/MS) analysis, we characterized 167 peptides belonging to 44 proteins, including gliadins, hordeins, and high- and low-molecular-weight glutenin subunits. Because of the interference from the overabundant yeast-derived peptides, we identified only a limited number of epitopes potentially triggering celiac disease. However, Weissbier samples contained 374, 372, and 382 ppm gliadin-equivalent peptides, as determined with the competitive G12 ELISA, which is roughly 10-fold higher than a lager BMB (41 ppm), thereby confirming that Weissbier is unsuited for celiacs. Western blot analysis demonstrated that Weissbier also contained large-sized prolamins immunoresponsive to antigliadin IgA antibodies from the pooled sera of celiac patients (n = 4). KEYWORDS: Weissbier, proteomics, peptidomics, celiac disease, IgE food allergens

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of the protein dynamic range (Proteominer),8 the proteomic methods have resulted in a practically complete inventory of beer proteins, which includes on the whole approximately 70 gene products deriving from barley (or adjuncts) and Saccharomyces spp. In general, proteins carry clear signs of process-induced modifications such as partial hydrolysis and glycation. Using LC-MS/MS methods, the beer peptidome has been characterized as well, though the record of beer peptides still appears partial because of the huge heterogeneity of the components.6,9,10 Several beer glutenlike peptides have been demonstrated harmful for celiacs using ex vivo assays.9,10 Nevertheless, when assayed by the R5 Ridascreen sandwich or competitive ELISA (Mendez antibody), a large part of the commercial lager and pale ale beers exhibited levels lower than 20 ppm of gluten equivalents,12,13 which corresponds to the threshold for a food to be labeled as gluten-free.14,15 Because of suppression effects, beer samples containing high levels of potentially harmful B hordeins exhibit a very low content of gluten equivalents when assayed by ELISA.10 A number of pitfalls related to the routinely carried out ELISA-based assays,16 with regard to the complexity of the

INTRODUCTION Proteins contribute to several quality traits of beer such as taste, texture, foam stability, and haze formation. The primary sources of beer proteins are barley (Hordeum vulgare) and, to a much lower extent, yeast and other cereals that are used for brewing (adjuncts). Barley contains celiacogenic gliadin-homologue prolamins (hordeins) in addition to several IgE-binding potential allergens that survive the malt proteases and the harsh brewing treatments. Prolamins are high-alcohol (60−70%) soluble proteins and are for the most part removed from the bulk solution during brewing. However, because of extensive proteolysis, a multitude of soluble peptides that may retain or even amplify the immune-stimulating potential is released into wort following the malting and mashing steps.1 Therefore, the characterization of both residual proteins and neoformed peptides has relevant immunological implications. During the past decade, several up-to-date proteomics strategies, including the classical 2D electrophoresis (2DE)− mass spectrometry (MS) and shotgun liquid chromatography− tandem MS (LC-MS/MS) approaches, have been exploited for the characterization of the beer proteome. Thus, barley Z4serpin, nonspecific lipid transfer proteins (nsLTPs), α-amylase/ trypsin inhibitors, and aveninlike protein A1 have been established as the dominant beer proteins.2−6 Combined with methods of prefractioning (OFFGEL)7 or with a compression © 2015 American Chemical Society

Received: Revised: Accepted: Published: 3579

February 2, 2015 March 5, 2015 March 20, 2015 March 20, 2015 DOI: 10.1021/acs.jafc.5b00631 J. Agric. Food Chem. 2015, 63, 3579−3586

Article

Journal of Agricultural and Food Chemistry

acid. Proteins were quantified using the Bradford assay and lyophilized. For the shotgun proteomic analysis, the protein fractions (>6 kDa) were digested with proteomic-grade modified trypsin (Sigma) at a 1:100 (w/w) enzyme−substrate ratio in 50 mM ammonium bicarbonate, pH 8.0, overnight at 37 °C. Peptides (6 kDa, here referred to as the protein fraction, were grossly separated from the low-molecular-weight (low-MW) peptides by SEC using G25 Econo-Pak 10 DG columns.9 Proteins from WB and BMB samples were analyzed with 1D SDS-PAGE, as shown in Figure 1. The main bands of WBs migrated with an apparent MW 3581

DOI: 10.1021/acs.jafc.5b00631 J. Agric. Food Chem. 2015, 63, 3579−3586

Article


Z-type serpins were by far the most represented proteins of WBs similar to those of the BMB.9 Z4 and Z7 serpins were from barley, five serpin proteoforms from wheat. By summing the relevant emPAI values, Z-serpins were estimated to occur at an expression degree comparable to one between those of barley and wheat. The search engine assigned three “unique” peptide matches to the barley ZX serpin, whose expression at the protein level has not been confirmed. These tryptic peptides actually were degenerate sequences that occur identical in both the Z4 and Z7 serpins. Therefore, ZX serpin was not computed within the list of the entries during the process of homology filtering. Similarly, several protein hits in Table 1 are indicative of a series of possible homologous gene products. Barley and wheat ns-LTPs, especially the ns-LTP1 types, ranked as the second-most abundant protein family of Weissbier. Despite the important proportion of malted wheat used for brewing, the barley ns-LTPs appeared much more abundant than those of wheat, according to the electrophoretic and emPAI data, probably because of several differences of the primary structures (barley and wheat ns-LTP1 and ns-LTP2 share 74 and 65% identity, respectively) that might affect the stability of the ns-LTPs to proteases.30 Anyhow, the proteins from both the sources are known to exhibit a relatively high resistance to proteases as well as to heat treatments. As a general trend, similar to BMB, the process of protein selection during Weissbier production enriches a few classes of endosperm proteins generally categorized among the seed pathogenesis-related proteins, which are able to survive even the harshest brewing injuries.31 Because of their functional role in the grain, pathogenesis-related proteins and protease inhibitors are also highly resistant to human physiological gastrointestinal digestion, and for this reason they could reach the gut in an immunologically active form and behave as type-I allergens in IgE-mediated food allergies.32 To this purpose, αamylases/trypsin inhibitors occurring as several isoforms in Weissbier are pest-resistance effectors besides being physiologically deputed to balance the accumulation and release of starch during the seed’s transition from dormancy to germination. αAmylases/trypsin inhibitors and thioredoxin are well established wheat IgE allergens, for instance in baker’s asthma. Recently, it has been proposed that α-amylases/trypsin inhibitors are stronger elicitors of the innate immune response and could be involved in several inflammatory disorders of the gastrointestinal tract, including the recently emerged nonceliac gluten sensitivity.33 Probably because of an effect of suppression exerted by yeast proteins, hordeins appeared to be missing in Weissbier, whereas in BMB they have been detected, albeit at low amounts. Minor levels of gluten proteins, including wheat α/β- and γ-gliadins as well as high- and low-MW glutenin subunits, were clearly identified instead, demonstrating that Weissbier do contain prolamins that are potentially harmful for celiacs. The relatively high number of gene products arising from S. cerevisiae (17 proteins, 29%) is consistent with the lack of the filtering step during brewing. Importantly, the great majority of the proteins identified are structurally and/or functionally related to components already described in BMB, thus actually validating the outcome of the analysis. Peptidome Analysis of Weissbier. The analysis by SEC demonstrated that 75−80% of the protein fraction of WBs consisted of medium-/small-sized soluble polypeptides released from the barley and wheat malt, similar to results from BMB

Figure 2. 2D electrophoresis (2DE) map of a representative Weissbier sample (WB#1). Major protein spots have been identified by PMF and on the basis of the MW/pI coordinates (also compare with ref 23).

tentatively assigned also relying on the MW/pI coordinates and by comparison with previous literature data.24,25 Apparently, the respective broad spots only contained the barley Z-serpins and ns-LTPs. However, the probable comigrating wheat homologue proteins were not detected due to the lower relative abundance and to a certain degree of dispersion over numerous isoforms, which are additionally affected by variable nonenzymatic glycation.26,27 Notably, a series of protein spots, previously described as α-amylase/trypsin inhibitors and aveninlike protein A1, was detected at MW = 13−17 kDa. Only protein traces possibly ascribable to hordeins/gliadins occurred at MW = 35−40 kDa and pI 7−7.5,2 whereas fragments of barley Z4-serpin and proteoforms of yeast triosephosphate isomerase populated the MW range = 28−40 kDa. Shotgun Proteomic Analysis of Weissbier. 2DE maps provided a general picture of the beer protein systems, evidencing the pattern of the main components and preserving the information about the protein integrity.28 Nevertheless, beer is a complex biochemical system constituted by a heterogeneous protein fraction and polypeptides with peculiar properties (e.g., very high pI) that are in part severely glycated, hydrolyzed, or aggregated.22 The Weissbier proteome is expectedly even more complex than the BMB one because of the occurrence of hexaploid wheat as an additional source of gene-expression products. In cases like this, gel-free shotgun proteomics (bottom-up strategy) expands the analytical dynamic range, contributing to comprehensive protein inventories that include those lessabundant components hardly detectable by different strategies.3,4,6,8,9 Thus, to deeply explore the proteome of Weissbier, the 2DE-based investigation was complemented with peptidecentric LC-ESI MS/MS-based shotgun proteomic analysis of a representative sample (WB#1). Overall, we identified 58 gene products with high confidence (p < 0.05), among which 41 (71%) were from H. vulgare and T. aestivum. The homologyfiltered protein hits are listed in Table 1. The emPAI value, assigned by the Mascot search engine on the basis of the spectral count in a label-free fashion,29 provided a rough estimate of the relative protein abundance. 3582


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Journal of Agricultural and Food Chemistry Table 1. Identification of Wheat Beer Proteins by the Shotgun Proteomic Approacha protein nameb

UNIPROT ID

ACC #c

MW (Da)/pI

emPAI

serpin-Z4 (H) serpin-Z1A (T) serpin-Z1B (T) serpin-Z2A (T) serpin-Z2B (T) serpin-Z1C (T) serpin-Z7 (H) serpin ZX (H)d ns-lipid transfer protein 1 (H) ns-lipid transfer protein 1 (T) ns-lipid transfer protein 2 (H) ns-lipid transfer protein 2 (T) glutenin high-MW subunit DY10 (T) glutenin high-MW subunit PW212 (T) glutenin low-MW subunit (T) α/β-gliadin A-IV (T) α/β-gliadin (T) γ-gliadin (T) γ-gliadin B (T) B1-hordein B3-hordein (fragment) γ3-hordein late embryogenesis abundant protein-group 3 (T) barwin (T) ABA-inducible protein PHV A1 (H) avenin like A2 (T) avenin like B2 (T) α-amylase inhibitor 0.19 (T) α-amylase inhibitor 0.28 (T) α-amylase/trypsin inhibitor CM2 (T) α-amylase/trypsin inhibitor CMb (H) α-amylase/trypsin inhibitor CM16 (T) trypsin inhibitor Cme (H) α-amylase/trypsin inhibitor CM1 (T) α-amylase/trypsin BMAI-1 α-amylase/trypsin inhibitor CMa (H) subtilisin-chymotrypsin inhibitor CI-1A (H) subtilisin-chymotrypsin inhibitor CI-1C (H) hordoindoline-B1 puroindoline-B oleosin 18 kDa (H)e 16.9 kDa class I heat shock protein 1 (S) glyceraldehyde-3-phosphate dehydrogenase 1 (S) glyceraldehyde-3-phosphate dehydrogenase 2 (S) glyceraldehyde-3-phosphate dehydrogenase 3 (S) enolase 1 (S) enolase 2 (S) cell wall mannoprotein PST1 (S) glucan 1,3-beta-glucosidase I/II (S) GPI-anchored protein YAR066W (S) triosephosphate isomerase (S) fructose-bisphosphate aldolase (S) putative protein YIL169C (S) thioredoxin-1 (S) thioredoxin-2 (S) phosphoglycerate mutase 1 (S) alcohol dehydrogenase 4 (S) pyruvate decarboxylase isozyme 1 (S)

SPZ4_HORVU SPZ1A_WHEAT SPZ1B_WHEAT SPZ2A_WHEAT SPZ2B_WHEAT SPZ1C_WHEAT BSZ7_HORVU SPZX_HORVU NLTP1_HORVU NLTP1_WHEAT NLT2_HORVU NLT2G_WHEAT GLT0_WHEAT GLT4_WHEAT GLTA_WHEAT GDA4_WHEAT GDA7_WHEAT GDB2_WHEAT GDBB_WHEAT HOR1_HORVU HOR3_HORVU HOG3_HORVU LEA3_WHEAT BARW_HORVU LEA1_HORVU AVLA2_WHEAT AVLB2_WHEAT IAA1_WHEAT IAA2_WHEAT IAAC2_WHEAT IAAB_HORVU IAC16_WHEAT IAAE_HORVU IAAC1_WHEAT IAA1_HORVU IAAA_HORVU ICIA_HORVU ICIC_HORVU HINB1_HORVU PUIB_WHEAT OLEO1_HORVU HS16A_WHEAT G3P1_YEAST G3P2_YEAST G3P3_YEAST ENO1_YEAST ENO2_YEAST PST1_YEAS7 EXG1_YEAST YA066_YEAST TPIS_YEAST ALF_YEAST YIQ9_YEAST TRX1_YEAST TRX2_YEAST PMG1_YEAST ADH4_YEAS7 PDC1_YEAST

P06293 Q41593 P93693 Q9ST57 P93692 Q9ST58 Q43492 Q40066 P07597 P24296 P20145 P82900 P10387 P08489 P10385 P04274 P04727 P08453 P06659 P06470 P06471 P80198 Q03968 P28814 P14928 P0CZ07 P0CZ05 P01085 P01083 P16851 P32936 P16159 P01086 P16850 P16968 P28041 P16062 P01054 Q9FSI9 Q10464 Q43769 P12810 P00360 P00358 P00359 P00924 P00925 Q12355 P23776 P0CX18 P00942 P14540 P40442 P22217 P22803 P00950 A6ZTT5 P06169

43277/5.7 43119/5.6 43033/5.4 43311/5.5 43011/5.2 42981/5.6 42821/5.4 42947/6.8 12301/8.7 11898/8.5 10357/6.8 9831/8.9 69629/7.6 89174/5.8 41020/9.0 34239/8.5 36118/8.6 37122/7.6 32967/8.1 33423/8.9 30196/7.7 31189/6.7 23238/8.8 13737/7.8 21820/9.0 19702/8.4 32514/7.8 13337/6.7 16800/7.4 15460/6.9 16526/5.8 15782/5.3 16135/7.5 15517/7.5 15816/5.4 15500/5.9 8882/5.2 8259/5.7 16122/8.7 16792/9.1 19495/9.7 16878/5.8 35750/8.3 35847/6.5 35747/6.5 46816/6.2 46915/5.7 45777/9.3 51311/4.6 20594/4.5 26796/5.7 39621/5.5 99737/4.4 11235/4.8 11204/4.8 26709/8.8 41084/5.9 61496/5.8

9.13 3.55 3.14 3.11 2.75 2.06 1.04 0.86 4.28 1.81 0.62 0.50 0.55 0.16 0.18 0.14 0.13 0.12 0.15 0.38 0.19 0.15 0.45 0.42 0.49 0.23 0.11 0.85 0.22 0.25 0.38 0.30 0.67 0.29 0.21 0.49 0.31 0.27 0.11 0.09 0.28 0.29 1.64 0.95 1.34 0.75 0.59 0.46 0.19 0.52 0.18 0.10 0.36 0.45 0.09 0.12 0.11 0.07

a

emPAI values provide a gross quantitative estimation.27 bH = H. vulgare; T = T. aestivum; S = S. cerevisiae. cUniProt accession number. dNot computed after homology filtering. ePeptide sequences (2) common to wheat oleosin. 3583


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additional epitope recognized by the R5,37 whereas the extended sequence contains the QXQPFP consensus sequence for antigliadin IgA and IgG binding.38−40 α-Gliadin fragment 33−54 (53−74 with the signal peptide) is part of toxic peptide 31−55, described as one of the main elicitors of the innate response in the immunopathogenesis of celiac disease.41 Epitope DQ2.5-glut-L142 is partly contained in the low-MW glutenin-derived peptides. Several other peptides of Weissbier harbored the restricted motifs associated with the induction of the celiac disease (e.g., PQQP).43,44 However, many additional extended multivalent harmful epitopes,36 distinctly sequenced in BMB,9 were missing in Weissbier. We argue that this apparent incongruence is due in part to the intrinsic system complexity and to the pitfalls of the peptidomic pipeline. Specifically, the relatively high abundance of yeast-derived peptides in WBs overwhelmed the gluten-derived peptides, which additionally suffer from intrinsic low ionization efficiency. The effect of suppression exerted by yeast-derived peptides is confirmed by the relatively scarce presence of fragments of Z-type serpin and ns-LTPs, which in contrast are largely represented in BMB.9 Also, it can not be ruled out that many gluten epitopes in Weissbier including the 33-mer are still encrypted within large-sized polypeptide fragments, so escaping MS/MS sequencing. To this purpose, it has to be underlined that several hundreds of peptide ions detected by MS remained unidentified. This demonstrates that the peptidomic picture emerging from the Weissbier system most likely is still the tip of an iceberg because the repertoire of peptide components is far from complete. G12 Competitive ELISA and Western Blotting of Weissbier. G12 competitive ELISA determinations supported the relatively high abundance of gluten epitopes in WBs. The reliability of the ELISA methods based on the Mendez R5 and Skerritt antibodies for quantifying hordeins in beer has been questioned.17 Moreover, it should be noted that the relationship between ELISA measurements of gluten and gluten toxicity is not clinically validated.45 In an attempt to overcome these shortcomings, an assay has been introduced that is based on the G12 antibody raised against hexapeptide epitope QPQLPY of the α-gliadin 33-mer.46 G12 antibody exhibits affinity to related toxic sequences from hydrolyzed prolamins of barley and rye and some cultivars of oats.47 Because the standard curve is built using variable concentrations of gliadins, the assay of substrates containing mixed glutenlike epitopes also derived from different cereal species such as barley, as in the case of Weissbier, could result in an overestimation of the results. However, specifically targeting epitopes reactive against T-cell of celiac patients, the G12 ELISA determinations appear to correlate with the actual immune reactivity of glutencontaining foods.11,48 The gluten content of Weissbier samples assayed with the competitive G12 ELISA was 374, 372, and 382 ppm (mean of three determinations with different beer batches; standard deviations < 5%) for WB#1, WB#2, and WB#3, respectively, which is almost 10-fold higher than that of the BMB sample (41 ppm). Gluten-free beer, used as the control, contained 2.8 ppm gliadin equivalents. Western blotting using a pool of sera from celiac patients as the source of antigliadin IgA antibodies confirmed the higher level of immunoreactive polypeptides in WB samples (Figure 4). Although the BMB sample only exhibited a responsive band because of a D-hordein,9 WBs showed complex patterns of

(Figure 3). The peptide fraction (MW < 6 kDa) from the WB samples showed indistinct RP-HPLC patterns containing a

Figure 3. Size-exclusion chromatography of polypeptides from a representative Weissbier sample (WB#1). Fractions were collected and analyzed by SDS-PAGE or by MALDI-TOF MS.

multitude of unresolved, low-abundance peaks and a few dominant peaks, attributed to free aromatic amino acids (e.g., tryptophan), detected at λ = 280 nm, and possibly to polyphenols (not shown).5 The low-MW peptide fraction of beer is extremely complex because of a series of concomitant factors, including the extreme heterogeneity of the parent proteins, which is further increased in Weissbier, the variable degree of heat-induced nonenzymatic glycation (Maillard reaction), and the unpredictable nature or lack of specificity of the proteolytic cleavage. The peptides from WB#1, representative of Weissbier samples, were analyzed by RPHPLC-ESI-Q-Orbitrap MS/MS analysis coupled to the database search. Relying on the high resolution of the MS (and MS/MS) measurements and the power of the search engine, we identified 167 peptides deriving from 44 different proteins (Table S1). Many of the peptides were from metabolic proteins of Saccharomyces spp., confirming that yeast contributes to the proteome and peptidome of WBs much more than it does to the peptidome of BMB. A significant number of peptides (37) arose from α/β- and γ-gliadins and several other from low- and high-MW glutenin subunits. Therefore, the overall immunogenic potential of Weissbier is clearly incompatible with a gluten-free diet. Some of the peptides could be encrypted in multiple protein entries because of the occurrence of repeated motifs within specific subfamilies of gluten proteins. In line with the parsimony principle, only protein entries representative of the subfamilies have been selected for cataloguing. The N-terminal region of α-gliadin that also is known to be resistant to proteolysis in sourdough34 was particularly well-represented. Interestingly, no peptides from ω-gliadins were detected, mirroring the lack of fragments from the homologue Chordeins in BMB.6,9 In contrast, significant amounts of Chordein-derived peptides have been recently described in a home-brewed beer.35 Immunogenic-proteases-resistant toxic 33-mer 56−88 of α2-gliadin36 was not detected, whereas Nterminal fragment 56−63 LQLQPFPQ of the same protein was clearly retrieved. (In Table S1 the sequence is indicated as 76− 83, because the numbering includes the 1−20 signal peptide.) This result indicates that the 33-mer α2-gliadin peptide is at least partly hydrolyzed during brewing. Pentapeptide QQPFP, common to other celiacogenic cereals and included in the 34−54 peptide of gliadins, is the main antigen of the R5 antibody used for assaying gluten in foods. QPFPQ, encrypted in the 56−63 α-gliadin fragment, is an 3584



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ASSOCIATED CONTENT

S Supporting Information *

Table S1, LC-ESI MS/MS-based identification of lowmolecular-weight peptides of wheat beer. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +39 0825 299218. Fax: +39 081 781585. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The sera of celiac patients were kindly gifted by Dr. Carmen Gianfrani who is gratefully acknowledged.

Figure 4. Western immunoblotting of Weissbier samples (WBs) compared to all-barley-malt (BMB) and gluten-free beers. SDS-PAGEseparated proteins were immunostained with pooled sera of celiac patients (n = 4) as the source of antigliadin IgA. Wheat (gliadins) and barley (hordeins) prolamins were used as the positive controls, whereas maize prolamins (zeins) were the negative control.

REFERENCES

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reactive components with the MW values expected for α/βgliadins (34−38 kDa) in addition to responsive high-MW glutenin subunits, which are D-hordein homologues. WBs also showed a smear at MW < 30 kDa indicative of possible immunoreactive large-sized gluten fragments. Consistent with the G12 evaluation, the Western blotting analysis demonstrated a potential immunotoxicity of WBs significantly higher than that of BMB. The much higher gluten level of WBs compared to that of the lager/Pilsner BMB also supports previous ELISA and Western blotting assessments carried out with firstgeneration antigluten antibodies.49

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CONCLUSIONS The proteome of Weissbier, here explored for the first time, has shown a protein pattern in which the wheat-derived gene products mirror the homologue barley-derived proteins already catalogued in BMB. The correspondence between barley- and wheat-derived proteins was not trivially predictable, taking into account that biochemical events occurring during wheat malting are largely underinvestigated compared to the barley counterpart50 and that wheat proteins significantly affect the properties of wheat beers.51 Because of the contribution of hexaploid wheat, the brewing grist of Weissbier and, consequently, its proteome are substantially different from those of BMB. Proteolysis during malting and other brewing steps releases an even more heterogeneous peptide assortment that has been partly characterized in this study. Comprehensive profiling of the protein/peptide repertory is partly hampered by the abundance of interfering yeast-derived gene products that are present in relatively large amounts as a consequence of the brewing process (Hefeweizen beer). However, it emerged clearly that Weissbier contains a large number of proteaseresistant and heat-stable proteins that are potential elicitors of IgE-mediated immunoreactions and sensitization events not related to celiac disease. The content of gluten epitopes in Weissbier, encrypted in either intact proteins or soluble hydrolyzed peptides, is much higher than that of lager/Pilsner BMB. The data presented herein demonstrate that although the compatibility of some lager BMB brands could be arguable13 Weissbier is definitely incompatible with the celiacs’ gluten-free diet. 3585


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Proteomics, Peptidomics, and Immunogenic Potential of Wheat Beer

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