Identification and analysis of multivalent proteolytically resistant peptides from gluten: implications for celiac sprue

Dietary gluten proteins from wheat, rye, and barley are the primary triggers for the immuno-pathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent molecular and structural analyses of representative gluten proteins, most notably alpha- and gamma-gliadin proteins from wheat, have improved our understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from alpha-gliadin under physiological conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here, we report three lines of investigation in support of this hypothesis. First, biochemical and immunological analysis of deletion mutants of alpha-2 gliadin confirmed that the DQ2 restricted T cell response to the alpha-2 gliadin are directed toward the epitopes clustered within the 33-mer. Second, proteolytic analysis of a representative gamma-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degradation, and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico analysis of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiologically generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome.     

A scaleable manufacturing process for pro-EP-B2, a cysteine protease from barley indicated for celiac sprue

Celiac Sprue is an inflammatory disease of the small intestine triggered by ingestion of dietary gluten, a family of glutamine and proline rich proteins found in common foodgrains such as wheat, rye, and barley. One potential therapy for this lifelong disease anticipates using an oral protease to detoxify gluten in vivo. Recent studies have shown that EP-B2 (endoprotease B, isoform 2) from barley is a promising example of such a glutenase, thus warranting its large-scale production for animal safety and human clinical studies. Here we describe a scaleable fermentation, refolding and purification process for the production of gram to kilogram quantities of pro-EP-B2 (zymogen form of EP-B2) in a lyophilized form. A fed-batch E. coli fermentation system was developed that yields 0.3-0.5 g purified recombinant protein per liter culture volume. Intracellular degradation of pro-EP-B2 during the fermentation was overcome by manipulating the fermentation temperature and duration of protein expression. A simple refolding protocol was developed using a fast dilution method to refold the enzyme at concentrations greater than 0.5 mg/mL. Kinetic analysis showed that pro-EP-B2 refolding is a first-order reaction with an estimated rate constant of 0.15 h(-1). A lyophilization procedure was developed that yielded protein with 85% recoverable activity after 7 weeks of storage at room temperature. The process was successfully scaled up to 100 L with comparable purity and recovery.     

High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: implications for celiac sprue.

Celiac Sprue is an HLA DQ2 (or DQ8)-associated autoimmune disorder of the human small intestine that is induced by dietary exposure to wheat gliadin and related proteins from barley, rye, and possibly other food grains. Recently, tissue transglutaminase (tTGase)-catalyzed deamidation of gliadin peptides has been shown to increase their potency for activating patient-derived, gliadin-specific T cells, suggesting that tTGase plays a causative role in the onset of an inflammatory response to toxic food grains. To dissect the molecular recognition features of tTGase for gluten derived peptides, the regioselectivity and steady-state kinetics of tTGase-catalyzed deamidation of known immunogenic peptides were investigated. The specificity of recombinant human tTGase for all immunogenic peptides tested was comparable to and, in some cases, appreciably higher than the specificity for its natural substrate. Although each peptide was glutamine-rich, tTGase exhibited a high degree of regioselectivity for a particular glutamine residue in each peptide. This selectivity correlated well with Q --> E substitutions that have earlier been shown to enhance the immunogenicity of the corresponding gliadin peptides. The specificity of tTGase toward homologues of PQPQLPY, a sequence motif found in immunodominant gliadin peptides, was analyzed in detail. Remarkably, the primary amino acid sequences of wheat-, rye-, and barley-derived proteins included many single-residue variants of this sequence that were high-affinity substrates of tTGase, whereas the closest homologues of this sequence found in rice, corn, or oat proteins were much poorer substrates of tTGase. (Rice, corn, and oats are nontoxic ingredients of the Celiac diet.) No consensus sequence for a high-affinity substrate of tTGase could be derived from our data, suggesting that the secondary structures of these food-grain peptides were important in their recognition by tTGase. Finally, under steady-state turnover conditions, a significant fraction of the tTGase active site was covalently bound to a representative high-affinity immunogenic gliadin peptide, suggesting a common mechanism by which cells responsible for immune surveillance of the intestinal tract recognize and generate an antibody response against both gliadin and tTGase. In addition to providing a quantitative framework for understanding the role of tTGase in Celiac Sprue, our results lay the groundwork for the design of small molecule mimetics of gliadin peptides as selective inhibitors of tTGase.     

Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ molecules

Celiac disease is caused by an abnormal intestinal T-cell response to gluten proteins of wheat, barley and rye. Over the last few years, a number of gluten T-cell epitopes restricted by celiac disease associated HLA-DQ molecules have been characterized. In this work, we give an overview of these epitopes and suggest a comprehensive, new nomenclature.     

Celiac disease: how complicated can it get?

In the small intestine of celiac disease patients, dietary wheat gluten and similar proteins in barley and rye trigger an inflammatory response. While strict adherence to a gluten-free diet induces full recovery in most patients, a small percentage of patients fail to recover. In a subset of these refractory celiac disease patients, an (aberrant) oligoclonal intraepithelial lymphocyte population develops into overt lymphoma. Celiac disease is strongly associated with HLA-DQ2 and/or HLA-DQ8, as both genotypes predispose for disease development. This association can be explained by the fact that gluten peptides can be presented in HLA-DQ2 and HLA-DQ8 molecules on antigen presenting cells. Gluten-specific CD4⁺ T cells in the lamina propria respond to these peptides, and this likely enhances cytotoxicity of intraepithelial lymphocytes against the intestinal epithelium. We propose a threshold model for the development of celiac disease, in which the efficiency of gluten presentation to CD4⁺ T cells determines the likelihood of developing celiac disease and its complications. Key factors that influence the efficiency of gluten presentation include: (1) the level of gluten intake, (2) the enzyme tissue transglutaminase 2 which modifies gluten into high affinity binding peptides for HLA-DQ2 and HLA-DQ8, (3) the HLA-DQ type, as HLA-DQ2 binds a wider range of gluten peptides than HLA-DQ8, (4) the gene dose of HLA-DQ2 and HLA-DQ8, and finally,(5) additional genetic polymorphisms that may influence T cell reactivity. This threshold model might also help to understand the development of refractory celiac disease and lymphoma.     

Identification of immunodominant epitopes of alpha-gliadin in HLA-DQ8 transgenic mice following oral immunization

Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant alpha-gliadin (r-alpha-gliadin) showing the most conserved sequence among the fraction of alpha-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-alpha-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-alpha-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120-139) and p23 (aa 220-239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-alpha-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-gamma secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.     

Circular dichroism and nuclear magnetic resonance spectroscopic analysis of immunogenic gluten peptides and their analogs

Celiac Sprue, or gluten-sensitive enteropathy, is an inheritable human disease of the small intestine that is triggered by the dietary intake of gluten. Recently, several Pro- and Gln-rich peptide sequences (most notably PQPQLPY and analogs) have been identified from gluten with potent immunogenic activity toward CD4(+) T cells from small intestinal biopsies of Celiac Sprue patients. These peptides have three unusual properties. First, they are relatively stable toward further proteolysis by gastric, pancreatic, and intestinal enzymes. Second, they are recognized and deamidated by human tissue transglutaminase (tTGase) with high selectivity. Third, tTGase-catalyzed deamidation enhances their affinity for HLA-DQ2, the disease-specific class II major histocompatibility complex heterodimer. In an attempt to seek a mechanistic explanation for these properties, we undertook secondary structural studies on PQPQLPY and its analogs. Circular dichroism studies on a series of monomeric and dimeric analogs revealed a strong polyproline II helical propensity in a subset of them. Two-dimensional nuclear magnetic resonance spectroscopic analysis confirmed a polyproline II conformation of PQPQLPY, and was also used to elucidate the secondary structure of the most helical variant, (D-P)QPQLPY. Remarkably, a strong correlation was observed between polyproline II content of naturally occurring gluten peptides and the specificity of human tTGase toward these substrates. Analogs with up to two D-amino acid residues retained both polyproline II helical content and transglutaminase affinity. Since the Michaelis constant (K(m)) is the principal determinant of tTGase specificity for naturally occurring gluten peptides and their analogs, our results suggest that the tTGase binding site may have a preference for polyproline II helical substrates. If so, these insights could be exploited for the design of selective small molecule inhibitors of this pharmacologically important enzyme.     

Recent advances in biosensors for diagnosis of celiac disease: A review

Celiac disease (CD) is an intestinal issue activated by the inappropriate immune reaction towards gluten protein of wheat, rye, barley, oats, and autoantigen, tissue transglutaminase. Regardless of the accessibility of immunochemical conventions for research facility analysis of CD, there is as yet a need of speedier, less expensive, and simpler devices for diagnosing CD. This review concentrates on progresses in biosensors for diagnosing CD in perspective of the scaled down hardware, multianalyte discovery and low sample volume necessity. Various recently developed biosensors in this field are presented.     

Medical nutrition therapy: use of sourdough lactic acid bacteria as a cell factory for delivering functional biomolecules and food ingredients in gluten free bread

Celiac disease (CD) is an immune-mediated disease, triggered in genetically susceptible individuals by ingesting gluten from wheat, rye, barley, and other closely related cereal grains. Currently, the estimated prevalence of CD is around 1 % of the population in the western world and medical nutritional therapy (MNT) is the only accepted treatment for celiac disease. To date, the replacement of gluten in bread presents a significant technological challenge for the cereal scientist due to the low baking performance of gluten free products (GF). The increasing demand by the consumer for high quality gluten-free (GF) bread, clean labels and natural products is rising. Sourdough has been used since ancient times for the production of rye and wheat bread, its universal usage can be attributed to the improved quality, nutritional properties and shelf life of sourdough based breads. Consequently, the exploitation of sourdough for the production of GF breads appears tempting. This review will highlight how sourdough LAB can be an efficient cell factory for delivering functional biomolecules and food ingredients to enhance the quality of gluten free bread.     

No genetic association of the human prolyl endopeptidase gene in the Dutch celiac disease population

Celiac disease (CD) is a complex genetic disorder of the small intestine. The DQ2/DQ8 human leucocyte antigen (HLA) genes explain approximately 40% of the genetic component of the disease, but the remaining non-HLA genes have not yet been identified. The key environmental factor known to be involved in the disease is gluten, a major protein present in wheat, barley, and rye. Integrating microarray data and linkage data from chromosome 6q21-22 revealed the prolyl endopeptidase (PREP) gene as a potential CD candidate in the Dutch population. Interestingly, this gene encodes for the only enzyme that is able to cleave the proline-rich gluten peptides. To investigate the role of the human PREP gene as a primary genetic factor in CD, we conducted gene expression, sequence analysis, and genetic association studies of the PREP gene and determined PREP enzyme activity in biopsies from CD patients and controls. Sequence analysis of the coding region of the PREP gene revealed two novel polymorphisms. Genetic association studies using two novel polymorphisms and three known PREP variants excluded a genetic association between PREP and CD. Determination of PREP activity revealed weak but significant differences between treated and untreated CD biopsies (P < 0.05). Our results from the association study indicate that PREP is not a causative gene for CD in the Dutch population. These are further supported by the activity determinations in which we observed no differences in PREP activity between CD patients and controls.     

Low‐gluten,nontransgenic wheat engineered with CRISPR/Cas9

Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The α‐gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33‐mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33‐mer in the α‐gliadin genes. Twenty‐one mutant lines were generated, all showing strong reduction in α‐gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene‐free lines were identified, and no off‐target mutations have been detected in any of the potential targets. The low‐gluten, transgene‐free wheat lines described here could be used to produce low‐gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.     

Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide

Background and Aims

Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied.

Methods

Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin.

Results

The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease.

Conclusions

The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.     

Regeneration potential of different wheat, rye and barley species in leaf explant culture

Regeneration potential of different wheat, rye and barley species in leaf explant culture. Comparative analysis of the induction ability of morphogenetic processes in vitro has been carried out in 16 wheat genotypes, 4 barley species and 6 rye genotypes. It has been shown that tetra- and hexaploid wheat species as well as wild barley species exhibited the highest embryogenic potential in the leaf explant culture while diploid wheat species and rye genotypes showed the lowest one. Genotypic dependence of processes of callus formation, induction of embryogenic calli and regeneration was revealed in the studied species.     

Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease

Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.     

Celiac sprue: correlation with murine T cell responses to wheat gliadin components

Celiac sprue is a disease in humans that is characterized by small intestinal mucosal injury and malabsorption. Dietary exposure to gliadin and similar proteins in rye and barley activates the disease in susceptible individuals. Celiac sprue appears to be the only disease with a marked HLA-association in which the proteins that activate the disease currently are well known. However, bread wheat gliadins are a complex mixture of proteins that contain at least 40 different components. In the present study we have purified the major gliadin components of Scout 66 wheat and used these proteins to examine murine T cell proliferative responses to gliadin. Differences in T cell proliferation stimulated by alpha-, beta-, gamma-, and omega-gliadins paralleled the known structural differences among these proteins. After priming with whole gliadin, the components that stimulated T cell proliferation were the same as those recognized to activate celiac sprue in humans. Studies with reduced and alkylated A-gliadin (i.e., S-methyl A-gliadin) suggested that epitopes determined by the native conformation of A-gliadin may be important in its interaction with T cells. By using three different A-gliadin peptides that span the entire molecule, T cell proliferative responses were shown to be stimulated predominantly by antigenic determinants on the NH2-terminal peptide.     

Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract

Background

Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes.

Methodology/Principal Findings

Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD₆₂₀ 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70–75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3–10).

Conclusion/Significance

While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides.     

Targeted modification of wheat grain protein to reduce the content of celiac causing epitopes

The prolamin peptides in wheat gluten and in the homologous storage proteins of barley and rye cause painful chronic erasure of microvilli of the small intestine epithelium in celiac patients. If untreated, it can lead to chronic diarrhea, abdominal distension, osteoporosis, weight-loss due to malabsorption of nutrients, and anemia. In addition to congenital cases, life-long exposure to gluten proteins in bread and pasta can also induce development of celiac sprue in adults. To date, the only effective treatment is life-long strict abstinence from the staple food grains. Complete exclusion of dietary gluten is, however, difficult due to use of wheat in many foods, incomplete labeling and social constraints. Thus, finding alternative therapies for this most common foodborne disease remained an active area of research, which has led to many suggestions in last few years. The pros and cons associated with these therapies were reviewed in the present communication. As different celiac patients are immunogenic to different members of the undigestible proline/glutamine rich peptides of ～149 gliadins and low molecular weight glutenin subunits as well as the six high molecular weight glutenin subunits, an exhaustive digestion of the immunogenic peptides in the stomach, duodenum, jejunum, and ileum of celiacs is required. In view of the above, we evaluated the capacity of cereal grains to synthesize and store the enzymes prolyl endopeptidase from Flavobacterium meningosepticum and the barley cysteine endoprotease B2, which in combination are capable of detoxifying immunogenic gluten peptides in a novel treatment of celiac disease.     

Proteomic analysis in allergy and intolerance to wheat products

Owing to its extensive use in the human diet, wheat is among the most common causes of food-related allergies and intolerances. Allergies to wheat are provoked by ingestion, inhalation or contact with either the soluble or the insoluble gluten proteins in wheat. Gluten proteins, and particularly the gliadin fraction, are also the main factor triggering celiac disease, a common enteropathy induced by ingestion of wheat gluten proteins and related prolamins from oat, rye and barley in genetically susceptible individuals. The role of gliadin and of its derived peptides in eliciting the adverse reactions in celiac disease are still far from being completely explained. Owing to its unique pathogenesis, celiac disease is widely investigated as a model immunogenetic disorder. The structural characterization of the injuring agents, the gluten proteins, assumes a particular significance in order to deepen the understanding of the events that trigger this and similar diseases at the molecular level. Recent developments in proteomics have provided an important contribution to the understanding of several basic aspects of wheat protein-related diseases. These include: the identification of gluten fractions and derived peptides involved in wheat allergy and intolerance, including celiac disease, and the elucidation of their mechanism of toxicity; the development and validation of sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for intolerant patients; and the formulation of completely new substitute foods and ingredients to replace the gluten-based ones. In this article, the main aspects of current and prospective applications of mass spectrometry and proteomic technologies to the structural characterization of gluten proteins and derived peptides are critically presented, with a focus on issues related to their detection, identification and quantification, which are relevant to the biochemical, immunological and toxicological aspects of wheat intolerance.     

Proteomic analysis in allergy and intolerance to wheat products

Owing to its extensive use in the human diet, wheat is among the most common causes of food-related allergies and intolerances. Allergies to wheat are provoked by ingestion, inhalation or contact with either the soluble or the insoluble gluten proteins in wheat. Gluten proteins, and particularly the gliadin fraction, are also the main factor triggering celiac disease, a common enteropathy induced by ingestion of wheat gluten proteins and related prolamins from oat, rye and barley in genetically susceptible individuals. The role of gliadin and of its derived peptides in eliciting the adverse reactions in celiac disease are still far from being completely explained. Owing to its unique pathogenesis, celiac disease is widely investigated as a model immunogenetic disorder. The structural characterization of the injuring agents, the gluten proteins, assumes a particular significance in order to deepen the understanding of the events that trigger this and similar diseases at the molecular level. Recent developments in proteomics have provided an important contribution to the understanding of several basic aspects of wheat protein-related diseases. These include: the identification of gluten fractions and derived peptides involved in wheat allergy and intolerance, including celiac disease, and the elucidation of their mechanism of toxicity; the development and validation of sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for intolerant patients; and the formulation of completely new substitute foods and ingredients to replace the gluten-based ones. In this article, the main aspects of current and prospective applications of mass spectrometry and proteomic technologies to the structural characterization of gluten proteins and derived peptides are critically presented, with a focus on issues related to their detection, identification and quantification, which are relevant to the biochemical, immunological and toxicological aspects of wheat intolerance.     

The purification and characterization of homologous high molecular weight storage proteins from grain of wheat,rye and barley

Summary Homologous high molecular weight storage prolamins were purified from grain of wheat, rye and barley using combinations of gel filtration, ion-exchange chromatography and preparative isoelectric focusing. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that the components were single bands with apparent mol.wts. of above 100,000. Molecular weights determined by sedimentation equilibrium ultracentrifugation were considerably lower; 54,700, 67,600 and 69,600 for the components from barley, rye and wheat respectively. Amino acid analysis showed the presence of 13.6 to 16.5 mol% glycine, 29.6 to 34.0 mol% glutamate + glutamine, 11.4 to 13.7 mol% proline and a total of 4.0 to 5.7 mol% basic amino acids. Automated N-terminal amino acid sequencing of the component from wheat showed the presence of cysteine residues at positions 5 and 10, and this is discussed in relation to the possible role of these proteins in the visco-elastic gluten network.