Proteomics offers one of the best approaches for the functional analysis of the genome, generating detailed information that can be integrated with that obtained by other classic and omics approaches .......
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The consumption of wheat, rye, and barley may cause adverse reactions to wheat such as celiac disease, non-celiac gluten/wheat sensitivity, or wheat allergy. The storage proteins (gluten) are known ...as major triggers, but also other functional protein groups such as α-amylase/trypsin-inhibitors or enzymes are possibly harmful for people suffering of adverse reactions to wheat. Gluten is widely used as a collective term for the complex protein mixture of wheat, rye or barley and can be subdivided into the following gluten protein types (GPTs): α-gliadins, γ-gliadins, ω5-gliadins, ω1,2-gliadins, high- and low-molecular-weight glutenin subunits of wheat, ω-secalins, high-molecular-weight secalins, γ-75k-secalins and γ-40k-secalins of rye, and C-hordeins, γ-hordeins, B-hordeins, and D-hordeins of barley. GPTs isolated from the flours are useful as reference materials for clinical studies, diagnostics or in food analyses and to elucidate disease mechanisms. A combined strategy of protein separation according to solubility followed by preparative reversed-phase high-performance liquid chromatography was employed to purify the GPTs according to hydrophobicity. Due to the heterogeneity of gluten proteins and their partly polymeric nature, it is a challenge to obtain highly purified GPTs with only one protein group. Therefore, it is essential to characterize and identify the proteins and their proportions in each GPT. In this study, the complexity of gluten from wheat, rye, and barley was demonstrated by identification of the individual proteins employing an undirected proteomics strategy involving liquid chromatography-tandem mass spectrometry of tryptic and chymotryptic hydrolysates of the GPTs. Different protein groups were obtained and the relative composition of the GPTs was revealed. Multiple reaction monitoring liquid chromatography-tandem mass spectrometry was used for the relative quantitation of the most abundant gluten proteins. These analyses also allowed the identification of known wheat allergens and celiac disease-active peptides. Combined with functional assays, these findings may shed light on the mechanisms of gluten/wheat-related disorders and may be useful to characterize reference materials for analytical or diagnostic assays more precisely.
The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought ...to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata B2, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC−MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5−Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.
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Barley is commonly used in malting and brewing, and spent grain is repurposed for other foods. Barley contains gluten proteins called hordeins that cause intestinal damage and disease symptoms if ...eaten by people with celiac disease and related conditions. While the mashing process in brewing can partially hydrolyze immunogenic epitopes in hordeins, the immunogenic epitope load between the starting malt and spent grain has not been investigated. Herein, we quantified hordeins in commercially available spent grain and from matching malt. Liquid chromatography–mass spectrometry (LC-MS) and sandwich and competitive R5 ELISAs were used for quantification, revealing a higher abundance of gluten proteins in the spent grain product compared with the input malt. Certain hordein subtypes were enriched while others were depleted, and overall protein content was higher in spent grain. This suggests that the mashing process selectively extracts nonprotein components, leaving protein and hordein content elevated in spent grain. The spent grain products tested were not safe for consumers with celiac disease.
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During brewing, gluten proteins may be solubilized, modified, complexed, hydrolyzed, and/or precipitate. Gluten fragments that persist in conventional beers render them unsuitable for people with ...celiac disease (CD) or gluten intolerance. Barley-based beers crafted to remove gluten using proprietary precipitation and/or application of enzymes, e.g. prolyl endopeptidases (PEP) that degrade the proline-rich gluten molecules, are available commercially. Gluten measurement in fermented products remains controversial. The industry standard, a competitive ELISA, may indicate gluten values <20 mg/kg, which is deemed safe for people with CD. However, in this study, liquid chromatography–mass spectrometry analyses revealed gluten peptides derived from hydrolyzed fragments, many >30 kDa in size. Barley gluten (hordeins) were detected in all beers analyzed with peptides representing all hordein classes detected in conventional beers but also, alarmingly, in many gluten-reduced beers. It is evident that PEP digestion was incomplete in several commercial beers, and peptides comprising missed cleavages were identified, warranting further optimization of PEP application in an industrial setting.
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Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed ...by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic.
We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS).
Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60-140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins.
ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique, enabling the quantification of individual hordein isoforms. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS.
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The trend of the number of publications on a research field is often used to quantify research interest and effort, but this measure is biased by general publication record inflation. This study ...introduces a novel metric as an unbiased and quantitative tool for trend analysis and bibliometrics. The metric was used to reanalyze reported publication trends and perform in-depth trend analyses on patent groups and a broad range of field in the life-sciences. The analyses confirmed that inflation bias frequently results in the incorrect identification of field-specific increased growth. It was shown that the metric enables a more detailed, quantitative and robust trend analysis of peer reviewed publications and patents. Some examples of the metric's uses are quantifying inflation-corrected growth in research regarding microplastics (51% ± 10%) between 2012 and 2018 and detecting inflation-corrected growth increase for transcriptomics and metabolomics compared to genomics and proteomics (Tukey post hoc p<0.0001). The developed trend-analysis tool removes inflation bias from bibliometric trend analyses. The metric improves evidence-driven decision-making regarding research effort investment and funding allocation.
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The efficiency of gluten extraction is of critical importance to the results derived from any analytical method for gluten detection and quantitation, whether it employs reagent-based technology ...(antibodies) or analytical instrumentation (mass spectrometry). If the target proteins are not efficiently extracted, the end result will be an under-estimation in the gluten content posing a health risk to people affected by conditions such as celiac disease (CD) and nonceliac gluten sensitivity (NCGS). Five different extraction protocols were investigated using LC-MRM-MS for their ability to efficiently and reproducibly extract gluten. The rapid and simple “IPA/DTT” protocol and related “two-step” protocol were enriched for gluten proteins, 55/86% (trypsin/chymotrypsin) and 41/68% of all protein identifications, respectively, with both methods showing high reproducibility (CV < 15%). When using multistep protocols, it was critical to examine all fractions, as coextraction of proteins occurred across fractions, with significant levels of proteins existing in unexpected fractions and not all proteins within a particular gluten class behaving the same.
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•An optimised protein extraction method was developed to extract ATIs from barley.•LC-MRM-MS analysis was used to compare extraction method efficiency.•A sequential two-step protocol consistently ...increased ATI yield (<12% variation).•The protocol was successfully applied to measure six ATIs across 12 barley cultivars.
Plant defense protein α-amylase trypsin inhibitors (ATIs) have been proposed as one of the triggers of non-coeliac gluten sensitivity, however there have been no focused studies on their optimal extraction and quantitation from cereal grains. The efficiency of extraction is of utmost interest for the downstream detection and characterisation. In the present study, three extraction buffers and two modified protocols were investigated using LC-MRM-MS in order to examine their ability to efficiently and repeatably extract ATIs from selected barley cultivars. Initially, three extraction buffers IPA/DTT, urea and Tris-HCl were used to extract ATIs from two selected barley cultivars, Commander and Hindmarsh. The results obtained from the preliminary study showed that IPA/DTT and urea-based buffer extraction could yield ∼70% and ∼45% more ATIs, respectively than a buffer based on Tris-HCl extraction, with all methods showing high repeatability (CV < 15%). A multi-step protocol, employing IPA/DTT and urea improved the extraction efficiency in comparison to the single buffer extraction protocols (p<0.0001). When solutions from parallel extractions using IPA/DTT and urea were combined, the results were comparable (p = 0.99) with a sequential multi-step IPA/DTT-urea protocol. However, the repeatability of the combined process was compromised, as discerned by greater variation (CV>30%). The optimised sequential two-step extraction protocol was successfully used to extract and quantify ATIs from 12 barley cultivars. LC–MS analysis revealed that cv Yagan and cv Scope contain the higher levels (∼143% relative to the average barley ATI content), whereas cultivars Fleet (61%), Baudin (77%) and Commander (79%) contained the lowest levels. The libraries of ATIs identified and the quantitative methods described here provide a foundation for the future application of MS-based quantitative methodologies to detect and quantify ATIs in barley varieties and in food products.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
10.
Perspectives on Future Protein Production Colgrave, Michelle L; Dominik, Sonja; Tobin, Aarti B ...
Journal of agricultural and food chemistry,
12/2021, Volume:
69, Issue:
50
Journal Article
Peer reviewed
Open access
An increasing world population, rising affluence, urbanization, and changing eating habits are all contributing to the diversification of protein production. Protein is a building block of life and ...is an essential part of a healthy diet, providing amino acids for growth and repair. The challenges and opportunities for production of protein-rich foods from animals (meat, dairy, and aquaculture), plant-based sources (pulses), and emerging protein sources (insects, yeast, and microalgae) are discussed against the backdrop of palatability, nutrition, and sustainability.
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