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- α-amylase/trypsin inhibitors (9)
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- wheat (4)
- Dormancy (3)
- Flower buds (3)
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The application of technical enzymes is a potential tool in modulating the dough and baking quality of cereal products. No endogenous amylases (alpha- and beta-forms) are present in mature wheat grains; they may be synthesized or activated during germination. Hence, microbial alpha-amylases are added to the dough, being resistant to the endogenous alpha-amylase/trypsin inhibitors. Here, we report on the initial identification of two technical enzymes from a commercial sample based on an in-gel tryptic digestion coupled with MALDI-MS analysis. The primary component of the protein fraction with 51.3 kDa was alpha-amylase from Aspergillus species. A second major protein with 24.8 kDa was identified as endo-1,4-xylanase from Thermomyces lanuginosus. In the following experimental work up, a targeted proteomics approach utilizing the combination of specific proteolytic digestion of the added amylase and xylanase in wheat flour, dough or baked products, solid phase extraction of released peptides and their detection using LC-MS/MS was optimized. The targeted (MRM) MS/MS peptide signals showed that the peptide "ALSSALHER" (MW = 983) originating from amylase and "GWNPGLNAR" (MW = 983) from xylanase can be used to identify the corresponding technical enzymes added. Consequently, locally available baked products were tested and found to contain these enzymes as supplementary ingredients. (C) 2014 Elsevier Ltd. All rights reserved.
Many biochemical processes are involved in regulating the consecutive transition of different phases of dormancy in sweet cherry buds. An evaluation based on a metabolic approach has, as yet, only been partly addressed. The aim of this work, therefore, was to determine which plant metabolites could serve as biomarkers for the different transitions in sweet cherry buds. The focus here was on those metabolites involved in oxidation-reduction processes during bud dormancy, as determined by targeted and untargeted mass spectrometry-based methods. The metabolites addressed included phenolic compounds, ascorbate/dehydroascorbate, reducing sugars, carotenoids and chlorophylls. The results demonstrate that the content of phenolic compounds decrease until the end of endodormancy. After a long period of constancy until the end of ecodormancy, a final phase of further decrease followed up to the phenophase open cluster. The main phenolic compounds were caffeoylquinic acids, coumaroylquinic acids and catechins, as well as quercetin and kaempferol derivatives. The data also support the protective role of ascorbate and glutathione in the para- and endodormancy phases. Consistent trends in the content of reducing sugars can be elucidated for the different phenophases of dormancy, too. The untargeted approach with principle component analysis (PCA) clearly differentiates the different timings of dormancy giving further valuable information.
Many biochemical processes are involved in regulating the consecutive transition of different phases of dormancy in sweet cherry buds. An evaluation based on a metabolic approach has, as yet, only been partly addressed. The aim of this work, therefore, was to determine which plant metabolites could serve as biomarkers for the different transitions in sweet cherry buds. The focus here was on those metabolites involved in oxidation-reduction processes during bud dormancy, as determined by targeted and untargeted mass spectrometry-based methods. The metabolites addressed included phenolic compounds, ascorbate/dehydroascorbate, reducing sugars, carotenoids and chlorophylls. The results demonstrate that the content of phenolic compounds decrease until the end of endodormancy. After a long period of constancy until the end of ecodormancy, a final phase of further decrease followed up to the phenophase open cluster. The main phenolic compounds were caffeoylquinic acids, coumaroylquinic acids and catechins, as well as quercetin and kaempferol derivatives. The data also support the protective role of ascorbate and glutathione in the para- and endodormancy phases. Consistent trends in the content of reducing sugars can be elucidated for the different phenophases of dormancy, too. The untargeted approach with principle component analysis (PCA) clearly differentiates the different timings of dormancy giving further valuable information.
The transition from dormant stage to the beginning of growth was first obvious by markedly changes of the water content. The phase from green tip to tight cluster, with a length of only 4 days, was the period of the most physiological activity in single buds, because of the highest daily accumulation rates of fresh/dry weight, C, N. We assume a concentration dependant regulation of the member of the aspartate family (asparagine, aspartic acid, isoleucine) during dormancy, growth and development in sweet cherry buds. The ABA content showed 2011/12 a clear bimodal pattern which was at lower level similar in 2012/13, but not so strong incisive. In both years, the first peak was probably related to the end of endodormancy. However the ABA-isomer content showed in both seasons a unimodal pattern. The maximum of the ratio of ABA-isomer/ABA indicated the beginning of ontogenetic development which starts 3 and 2 weeks later, respectively. Our results suggest that ABA and the ABA-isomer in the sweet cherry buds regulate differentiated metabolic processes in the dormant stage and during bud growth and development. After replication in the season 2013/14 the estimated dates of release of endodormancy, beginning of ecodormancy and start of ontogenetic development will be used to validate and improve phenological models for the beginning of cherry blossom. (C) 2014 Elsevier B.V. All rights reserved.
Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC–MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.
Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC–MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.
The right choice of analytical methods for plant allergen quantification is a deciding factor for the correct assessment and labeling of allergens in processed food in view of consumer protection. The aim of the present study was to develop a validated target peptide multi-method by LC/MS/MS providing high specificity and sensitivity for plant allergen protein detection, plant identification in vegan or vegetarian products using peptide markers for quantification. The methodical concept considers the selection of target peptides of thermostable allergenic plant proteins (Gly m6 soy, Ses i6 sesame and (beta-conglutin from white lupine) by data base research, BLAST and in silico digestion using Skyline software. Different allergenic concentration levels of these proteins were integrated into our own reference bakery products and quantified with. synthesized isotopically labeled peptides after in-solution digestion using LC/MS/MS. Recovery rates within the range of 70-113% and LOQ of 10 ppm-50 ppm (mg allergenic food/kg) could be determined. The results are independent of thermal processing applied during baking and of epitope binding site for the tested allergens. (C) 2016 Elsevier Ltd. All rights reserved.
Identification and LC-MS/MS-based analyses of technical enzymes in wheat flour and baked products
(2016)
The use of technical enzymes in bakery industry is necessary for a consistent and good quality of baked products. Since the cultivation of cereals leads to low amounts of endogenous enzymes being present, a need of their commercial alternatives is becoming a routine process in order to meet the consumer quality demands. Targeted quantification proteomics-based methods are necessary for their detection to meet the regulatory criteria. Here, we initially report on the identification of Lipase FE-01, a lipase from fungus Thermomyces lanuginosus, as analyzed by SDS-PAGE, in-Gel digestion, and MALDI-TOF-MS. In further experiments, the focus of the study was directed toward an extensive use and optimization of in-solution enzymatic digestion in combination with LC-MS/MS techniques in identification of specific peptide markers and finally in utilization of the latter in delivering reproducible quantification data for several different technical enzymes (alpha-amylases, xylanase, and lipases from microbial origin) in complex matrices such as baked bread and wheat flour. Two digestion protocols (a fast option using thermocycler program and the well-established overnight method) were tested, and both of these can be successfully applied. The application of isotopically labeled analogs of the MRM targeted peptides as internal standards and the addition of an internal protein standard during the extraction/digestion experiment were compared to determine the optimal quantification algorithm of the recovered enzyme concentrations. Thus, a standardized sensitive LC-MS/MS method could be developed to determine technical enzymes as forthcoming ingredients in the prefabricated food formulations in concentrations lower than 10 ppm.
The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.
The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.