Mass spectrometry analysis of gliadins in celiac disease

In recent years, scientific research on wheat gluten proteins has followed three main directions aimed at (1) finding relationships between individual genetic alleles coding for gliadins, high or low molecular weight glutenin subunits, and the viscoelastic dough properties of flour-derived products such as pasta and bread; (2) identifying prolamins and derived peptides involved in celiac disease, a pathological condition in which the small intestine of genetically predisposed individuals is reversibly damaged; and (3) developing and validating sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for celiac disease patients. In this review, the main aspects of current and perspective applications of mass spectrometry and proteomic technologies to the structural characterization of gliadins are presented, with focus on issues related to detection, identification, and quantification of intact gliadins, as well as gliadin-derived peptides relevant to the biochemical, immunological, and toxicological aspects of celiac disease.     

Potential of using plant proteins and chicken feathers for cotton warp sizing

Two plant proteins, soyprotein and wheat gluten, and chicken feathers used to size cotton substrates provided sizing performance similar to starch and were also easily degraded in activated sludge. Sizing is an essential process to impart protection to warp yarns and increase weaving efficiency. Cotton yarns have traditionally been sized with starch, modified starch derivatives, CMC, poly vinyl alcohol (PVA), or a combination thereof along with quite a few other fiber binding ingredients. Although starch and starch derivatives are extensively used for sizing, there can be several limitations including less-than-satisfactory sizing performance and difficulties in desizing starch based size. Plant proteins such as wheat gluten, soyproteins and poultry feathers are available in large quantities at low cost and have limited industrial applications. However, these proteins are known to have excellent film-forming properties, a primary requirement for a warp size, and have also been used as adhesives. Using proteins as warp sizing agents on cotton yarns potentially could provide acceptable sizing performance and be cost-effective, as well. In this research, soyproteins, wheat gluten, and chicken feathers were studied for exploring their feasibility for sizing, desizing, biodegradability, and ability to replace starch and PVA for sizing cotton yarns. It was found that all three proteins provided similar cohesion to fibers and abrasion resistance compared to starch. Protein sizes had significantly high BOD₅/COD ratio compared to PVA, suggesting that the proteins are easily degradable in textile effluent treatment plants.     

Preparation and characterization of enzymatically hydrolyzed prolamins from wheat,rye, and barley as references for the immunochemical quantitation of partially hydrolyzed gluten

Celiac disease (CD) is a permanent gastrointestinal disorder characterized by the intolerance to a group of proteins called gluten present in wheat, rye, barley, and possibly oats. The only therapy is a strict lifelong gluten-free diet. The standard method for gluten determination in foods produced for CD patients is the R5-enzyme-linked immunosorbent assay (ELISA) as proposed by the recent Codex Alimentarius Draft Revised Standard. This test is based on the determination of prolamins, the alcohol-soluble proteins of gluten, and is available as a sandwich ELISA for intact proteins and as a competitive ELISA for gluten-derived peptides. While the suitability of the sandwich ELISA including a wheat prolamin (gliadin) reference for calibration has been shown by various studies and a ring test, the competitive ELISA still lacks a convenient reference for the quantitation of gluten peptides in fermented cereal foods (e.g., sourdough products, starch syrup, malt extracts, beer). Therefore, the aim of the present study was to prepare a suitable reference for the quantitation of partially hydrolyzed gluten in fermented wheat, rye, and barley products. The prolamin fractions from barley (hordein) and rye (secalin) were isolated from corresponding flours by means of a modified preparative Osborne fractionation. The prolamin fraction from wheat was obtained as reference gliadin from the Prolamin Working Group. The prolamin fractions were successively digested by pepsin and trypsin or pepsin and chymotrypsin procedures, which have been used for CD-specific toxicity tests on cereal storage proteins for many years. The protein/peptide content (N × 5.7) of the prolamin fractions and digests, which was the basis for the calculation of the gluten content by means of ELISA, varied between 67.1% and 96.0%. The prolamin fractions and enzymatic digests were then tested for their response in both sandwich and competitive assays. Intact prolamins responded similarly in both ELISA showing no important differences between the cereals. In the case of digested proteins, however, the sandwich ELISA was considerably less sensitive than the competitive ELISA. The former provided approximately 40% and the latter 70% of the signal intensity obtained with the intact prolamins. Thus, the combination of the competitive ELISA and the enzymatic digests of prolamin fractions as reference was considered to be an adequate system for the analysis of partially hydrolyzed gluten. The limit of detection using a peptic-tryptic hordein digest as reference was 2.3 μg prolamin equivalent per milliliter, and the limit of quantitation was 6.7 μg prolamin equivalent per milliliter. This system was applied for the determination of gluten equivalents in five commercial beverages based on fermented cereals.      

FT-Raman Spectroscopy as a Tool to Study the Secondary Structures of Wheat Gliadin Proteins

Raman spectroscopy is a useful method in biological, biomedical, food, and agricultural studies, allowing the simultaneous examination of various chemical compounds and evaluation of molecular changes occurring in tested objects. The purpose of our research was to explain how the elimination of ω-fractions from the wheat gliadin complex influences the secondary structures of the remaining αβγ-gliadins. To this aim, we analyzed the endosperm of wheat kernels as well as gliadin proteins extracted from two winter wheat genotypes: wasko.gl+ (control genotype containing the full set of gliadins) and wasko.gl− (modified genotype lacking all ω-gliadins). Based on the decomposition of the amide I band, we observed a moderate increase in β-forms (sheets and turns) at the expense of α-helical and random coil structures for gliadins isolated from the flour of the wasko.gl− line. Since ω-gliadins contain no cysteine residues, they do not participate in the formation of the disulfide bridges that stabilize the protein structure. However, they can interact with other proteins via weak, low-energetic hydrogen bonds. We conclude that the elimination of ω-fractions from the gliadin complex causes minor modifications in secondary structures of the remaining gliadin proteins. In our opinion, these small, structural changes of proteins may lead to alterations in gliadin allergenicity.     

Use of capillary electrophoresis to monitor wheat maturation

Summary The maturation of wheat varieties with different harvest times has been examined by high-performance capillary electrophoresis. The unique proteins of the albumin, gliadin and glutenin fractions of Hungarian winter wheat cultivars Bánkúti 1201 (early harvest time), Martonvásári 23 (medium harvest time), and Martonvásári 15 (semi-late harvest time) were analysed. An acidic phosphate buffer containing a polymeric additive and organic modifiers was used in capillary zone electrophoresis mode. Formation of albumin followed the same time scale, and the patterns were quite similar, for all three cultivars. For gliadins and glutenins the time scale and patterns were different and some correlation was observed between harvest time and gliadin formation. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Nano-mechanical properties of starch and gluten biopolymers from atomic force microscopy

An original method based on atomic force microscopy (AFM) in contact mode was developed to abrade progressively the surface of tablets made of starch or gluten polymers isolated from wheat. The volume of the material removed by the tip was estimated from the analysis of successive topographic images of the surface, and the shear force was measured by keeping a constant normal force. Our data together with a simple tribological model provide clear evidence for a higher hardness and shear strength of starch compared to gluten. Gluten appears to have mechanical properties close to soft materials, such as talc, whereas starch displays higher hardness close to calcite. Our results are in a better agreement with structural properties of gluten (complex protein network) and starch (granular and semi-cristalline structure) than earlier studies by micro-indentation. This work shows that the AFM scratching method is relevant for the characterization of any polymer surface, in particular in application to materials made of different polymers at the nano-scale.     

Qualitative and quantitative analysis of wheat gluten proteins by liquid chromatography and electrospray mass spectrometry

Based on analysis by liquid chromatography/electrospray ionisation mass spectrometry, we have developed a new method for fast and sensitive fingerprinting of gliadins and glutenins in wheat flour. Using this procedure the two protein fractions from seven durum wheat varieties have been analysed by high resolution high performance liquid chromatographic separation coupled to accurate determination of molecular mass. In this way, the molecular mass of the single components from both gliadin and glutenin fractions were measured and more than forty components were detected for each fraction indicating a high heterogeneity. Although the chromatographic profiles were similar, the molecular masses of protein components with similar retention times among the varieties were often different. The difference ranged from a few mass units corresponding to single amino acid substitution(s) up to thousands implying peptide deletion or insertion along the protein chain. Two components representing about a half of the gliadin fraction, e.g. gamma(2)- and gamma(3)-gliadin, were identified through the N-terminal sequence and molecular mass determination. We suggest the use of the high level and the molecular mass of these gliadin components as markers to detect traces of wheat in gluten-free food preparations for celiac patients.     

Thermal transitions of gluten-free doughs as affected by water, egg white and hydroxypropylmethylcellulose

The thermal performance of a gluten-free bread dough consisting of a blend of non allergenic corn and cassava starches (75:25) with hydroxypropylmethylcellulose (HPMC) as gluten mimetic hydrocolloid in conjunction with egg white (EW) was determined by differential scanning calorimetry. In order to analyse the effects of different levels of the components (water: 80-110%, HPMC: 0-2% and EW: 0-10% over the starch blend) on the thermal transitions of the dough, a Doehlert design and a response surface methodology were used.The analysis of variance showed that EW did not affect the onset temperature of gelatinisation and HPMC did not affect the peak and conclusion temperatures. HPMC-water interactions mainly controlled the onset temperature of starch gelatinisation. On the other hand, the peak and conclusion temperatures were determined by the additive and opposite effects of water and EW.     

Starch Gelatinization Kinetics in Bread Dough. DSC investigations on 'simulated' baking processes

Starch gelatinization in wheat flour dough of various moisture contents was quantitatively evaluated by means of DSC. The experimental records were worked out in the form of excess heat capacity vs. T traces which were deconvoluted to single out the contribution of starch gelatinization from that of the decomposition of amylose-lipid complexes. The quantitative procedure used put into evidence that a third endothermic process would take place in the dough with a poor moisture content. DSC runs carried out with sealed pans (i.e., at constant moisture level) and open pans (from which some water was free to evaporate) allow simulation of two extreme conditions of a real baking process, namely that relevant to the central core and to superficial layer of a dough loaf, respectively. The extent of starch gelatinization occurred in these conditions was quantitatively assessed. These data were collected at various heating rates and used to define temperature-time-transformation(TTT) diagrams which are useful tools to predict the progress of baking for any given thermal history of the system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Electrophoresis and chromatography of wheat proteins: available methods, and procedures for statistical evaluation of the data.

Analysis of gluten proteins from the wheat grain endosperm has long challenged the analytical chemist. Several hundred unique polypeptides are present, many in large polymers. This complexity, plus useful relationships of composition to genotype and quality, encouraged development and application of electrophoresis and chromatography for gluten analysis. We review the methods of polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and high-performance liquid chromatography available for study of wheat proteins. Singly and in combination, they provide rapid, reproducible, high-resolution separations based on size, charge, or surface hydrophobicity. As challenging and important as the analyses themselves, however, is interpretation of data. Subjective evaluation is sometimes possible, but statistical methods such as similarity scores, clustering, principal components, multiple linear regression, and partial least squares now are increasingly used for data analysis. We review the use of these procedures, and precautions necessary to avoid misinterpretation of data. Optimal evaluation of protein analytical data will enhance the value of such analyses in wheat breeding, marketing, and processing.     

How the biodegradability of wheat gluten-based agromaterial can be modulated by adding nanoclays

The objective of this work was to investigate the influence of clay nanoparticles on the biodegradability of wheat gluten-based materials through a better understanding of multi-scale relationships between biodegradability, water transfer properties and structure of wheat gluten/clay materials. Wheat gluten/clay (nano)composites materials were prepared via bi-vis extrusion by using an unmodified sodium montmorillonite (MMT) and an organically modified MMT. Respirometric experiments showed that the rate of biodegradation of wheat gluten-based materials could be slowed down by adding unmodified MMT (HPS) without affecting the final biodegradation level whereas the presence of an organically modified MMT (C30B) did not significantly influence the biodegradation pattern. Based on the evaluation of the water sensitivity and a multi-scale characterization of material structure, three hypotheses have been proposed to account for the underlying mechanisms. The molecular/macromolecular affinity between the clay layers and the wheat gluten matrix, i.e. the ability of both components to establish interactions appeared as the key parameter governing the nanostructure, the water sensitivity and, as a result, the overall biodegradation process.     

Mass spectrometry in the characterization of cereal seed proteins

In less then a decade, applications of matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) mass spectrometry to the investigation of prolamins have rapidly evolved from measurements of the molecular mass of isolated proteins to a proteomic approach attempting to characterise the complete protein pattern in the seed. Mass spectrometry is currently making significant contributions to the understanding of the composition and structure of the gluten proteins and, in turn, to the elucidation of structure-function relationships. Results obtained using mass spectrometry, including determination of the molecular masses of prolamins, direct verification of gene-derived sequences, determination of the number of cysteine residues and localisation of disulphide bonds, investigation of the gluten toxicity for celiac patients, qualitative and quantitative determination of gliadins in food and determination of the protein pattern and its modification during seed maturation by proteomic approaches, are summarised here, to illustrate current trends and individuate possible future perspectives.     

Calorimetric and rheological properties of wheat flour suspensions and doughs: Effects of wheat types and milling procedure

Three types of wheat were submitted to two different milling procedures, giving rise to six flours which differed by some physico-chemical characteristics such as particle size, level of damaged starch and protein content. Differential scanning calorimetry was used for monitoring heat-induced structural changes in flour aqueous dispersions 80% water and in doughs 45% water. Differences between the thermal behaviour of the flour dispersions and doughs were explained mainly by differences in protein content. This result was confirmed after partial substitution of flour by gluten. Dynamic mechanical analysis performed at 20°C on the flour doughs indicated, as expected, a linear increase in the elastic modulus with increasing protein content. The results did not bring any evidence that, under these experimental conditions, starch damage might affect gluten hydration.This revised version was published online in November 2005 with corrections to the Cover Date.     

Separation of the unique proteins of wheat protein fractions by capillary electrophoresis

Summary The wheat maturation process was monitored by high-performance capillary electrophoresis. The different protein components of the albumin, globulin, gliadin and glutenin fractions from the Osborne extraction procedure were analysed. The wheat sample was a Hungarian winter wheat, cultivar Martonvásári 23. The protein fractions were analysed by capillary zone electrophoresis using a low pH phosphate buffer containing a polymeric additive and organic modifiers. The albumins and gliadins as well as glutenins showed a characteristic pattern of development during the maturation process. For these fractions the development occurred at different stages of maturation. The formation of protein fractions of wheat at different stages of maturation—and thus the entire maturation process—could be well characterised by high-performance capillary electrophoresis. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September, 1–3, 1999     

GC-MS analytical methods for the determination of personal-care products in water matrices

This article discusses the more recent methods combining gas chromatography and mass spectrometry (GC-MS) for analysis of personal-care products (PCPs) in water matrices. We describe different procedures for sample extraction and preparation as well as different instrumental methods commonly used for these compounds. GC-MS and GC-tandem MS (GC-MS²), which are complementary to liquid chromatography combined with MS (LC-MS), allow identification and quantification of PCPs belonging to different classes with the sensitivity and the selectivity necessary for environmental monitoring. The compounds investigated include fragrances (e.g., nitro and polycyclic musks), antimicrobial compounds (e.g., triclosan), ultraviolet blockers (e.g., methylbenzylidene camphor), antioxidants and preservatives (e.g., phenols and p-hydroxybenzoic acid (parabens)) and insect repellents (e.g., N,N-diethyl-m-toluamide (DEET)). We critically review data in the literature by focusing attention on analytical methods devoted to simultaneous detection and quantification of structurally diverse pharmaceuticals and PCPs.     

The Effect of α-, β- and γ-Cyclodextrin on Wheat Dough and Bread Properties

Cyclodextrins (CDs) are cyclic oligosaccharides that have found widespread application in numerous fields. CDs have revealed a number of various health benefits, making them potentially useful food supplements and nutraceuticals. In this study, the impact of α-, β-, and γ-CD at different concentrations (up to 8% of the flour weight) on the wheat dough and bread properties were investigated. The impact on dough properties was assessed by alveograph analysis, and it was found that especially β-CD affected the viscoelastic properties. This behavior correlates well with a direct interaction of the CDs with the proteins of the gluten network. The impact on bread volume and bread staling was also assessed. The bread volume was in general not significantly affected by the addition of up to 4% CD, except for 4% α-CD, which slightly increased the bread volume. Larger concentrations of CDs lead to decreasing bread volumes. Bread staling was investigated by texture analysis and low field nuclear magnetic resonance spectroscopy (LF-NMR) measurements, and no effect of the addition of CDs on the staling was observed. Up to 4% CD can, therefore, be added to wheat bread with only minor effects on the dough and bread properties.     

Characterizing in situ starch gelatinization

Data obtained by dynamic mechanical analysis and DSC analysis of durum wheat dough are presented and discussed. Doughs with water contents ranging from 45 to 55% (w/w) were subjected to sinusoidal shearing by means of a dynamic mechanical spectrometer (Rheometrics, RFS2) equipped with parallel plate geometry, 0.1 strain amplitude and 1 rad/min frequency. The tests were carried on in temperature sweep mode at a heating rate of 2°C min^–1.Wheat samples with water contents in the range between 7.5 and 37.5% and doughs with 37.5% moisture content were mixed for different times and subjected to DSC analysis (Perkin-Elmer, DSC-7) at a heating rate of 20°C min^–1.Dynamic mechanical analysis revealed that the relationship between the dynamic properties of the dough and the temperature was modified as the water content of the dough increased and was quite different from that for gluten.A similar response was observed in the course of temperature scans made by means of DSC. These experimental findings suggest that the water-starch interaction in the presence of a protein matrix is affected by the availability of water and that the protein system is a competitor with respect to starch.This work was supported by C. N. R. Progetto Strategico) Dieta Mediterranea.     

Portable single-sided NMR measurements at variable temperatures: Implementation of a thermo-controlled device and application to the heating of bread dough

A temperature control unit was implemented to vary the temperature of samples studied on a commercial Mobile Universal Surface Explorer nuclear magnetic resonance (MOUSE-NMR) apparatus. The device was miniaturized to fit the maximum MOUSE sampling depth (25 mm). It was constituted by a sample holder sandwiched between two heat exchangers placed below and above the sample. Air was chosen as the fluid to control the temperature at the bottom of the sample, at the interface between the NMR probe and the sample holder, in order to gain space. The upper surface of the sample was regulated by the circulation of water inside a second heat exchanger placed above the sample holder. The feasibility of using such a device was demonstrated first on pure water and then on several samples of bread dough with different water contents. For this, T1 relaxation times were measured at various temperatures and depths and were then compared with those acquired with a conventional compact closed-magnet spectrometer. Discussion of results was based on biochemical transformations in bread dough (starch gelatinization and gluten heat denaturation). It was demonstrated that, within a certain water level range, and because of the low magnetic field strength of the MOUSE, a linear relationship could be established between T1 relaxation times and the local temperature in the dough sample.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Ancient Wheat and Quinoa Flours as Ingredients for Pasta Dough—Evaluation of Thermal and Rheological Properties

The aim of this study was to investigate thermal and rheological properties of selected ancient grain flours and to evaluate rheological properties of mixtures thereof represented by pasta dough and dry pasta. Flours from spelt, einkorn, and emmer ancient wheat varieties were combined with quinoa flour. All these flour sources are considered healthy grains of high bioactive component content. Research results were compared to durum wheat flour or spelt wheat flour systems. Differential scanning calorimeter (DSC) and a rapid visco analyzer (RVA) were used to investigate the phase transition behavior of the flours and pasting characteristics of the flours and dried pasta. Angular frequency sweep experiments and creep and recovery tests of the pasta dough were performed. The main components modifying the pasta dough structure were starch and water. Moreover, the proportion of the individual flours influenced the rheological properties of the dough. The durum wheat dough was characterized by the lowest values of the K′ and K″ parameters of the power law models (24,861 Pa·sⁿ′ and 10,687 Pa·sⁿ″, respectively) and the highest values of the instantaneous (J₀) and retardation (J₁) compliances (0.453 × 10⁻⁴ Pa and 0.644 × 10⁻⁴ Pa, respectively). Replacing the spelt wheat flour with the other ancient wheat flours and quinoa flour increased the proportion of elastic properties and decreased values of the J₀ and J₁ of the pasta dough. Presence of the quinoa flour increased pasting temperature (from 81.4 up to 83.3 °C) and significantly influenced pasting viscosities of the spelt wheat pasta samples. This study indicates a potential for using mixtures of spelt, einkorn, and emmer wheat flours with quinoa flour in the production of innovative pasta dough and pasta products.