TY - JOUR A1 - Silva, Bibiana A1 - Oliveira Costa, Ana Carolina A1 - Tchewonpi, Sorel Sagu A1 - Bönick, Josephine A1 - Huschek, Gerd A1 - Gonzaga, Luciano Valdemiro A1 - Fett, Roseane A1 - Baldermann, Susanne A1 - Rawel, Harshadrai Manilal T1 - Comparative quantification and differentiation of bracatinga (Mimosa scabrella Bentham) honeydew honey proteins using targeted peptide markers identified by high-resolution mass spectrometry JF - Food research international N2 - Honey traceability is an important topic, especially for honeydew honeys, due to the increased incidence of adulteration. This study aimed to establish specific markers to quantify proteins in honey. A proteomics strategy to identify marker peptides from bracatinga honeydew honey was therefore developed. The proteomics approach was based on initial untargeted identification of honey proteins and peptides by LC-ESI-Triple-TOF-MS/MS, which identified the major royal jelly proteins (MRJP) presence. Afterwards, the peptides were selected by the in silico digestion. The marker peptides were quantified by the developed targeted LC-QqQ-MS/MS method, which provided good linearity and specificity, besides recoveries between 92 and 100% to quantify peptides from bracatinga honeydew honey. The uniqueness and high response in mass spectrometry were backed by further complementary protein analysis (SDS-PAGE). The selected marker peptides EALPHVPIFDR (MRJP 1), ILGANVK (MRJP 2), TFVTIER (MRJP 3), QNIDVVAR (MRJP 4), FINNDYNFNEVNFR (MRJP 5) and LLQPYPDWSWTK (MRJP 7), quantified by LC-QqQ-MS/MS, highlighted that the content of QNIDVVAR from MRJP 4 could be used to differentiate bracatinga honeydew honey from floral honeys (p < 0.05) as a potential marker for its authentication. Finally, principal components analysis highlighted the QNIDVVAR content as a good descriptor of the analyzed bracatinga honeydew honey samples. KW - Honeydew honey KW - Major royal jelly proteins KW - Marker peptides KW - High-resolution mass spectrometry KW - Principal component analysis Y1 - 2020 U6 - https://doi.org/10.1016/j.foodres.2020.109991 SN - 0963-9969 SN - 1873-7145 VL - 141 PB - Elsevier CY - New York, NY [u.a.] ER - TY - GEN A1 - Sagu Tchewonpi, Sorel A1 - Huschek, Gerd A1 - Waldbach Braga, Tess A1 - Rackiewicz, Michal A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.) T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1260 KW - wheat KW - α-amylase/trypsin inhibitors KW - fractionation KW - purification KW - reversed-phase chromatography KW - ion-exchange chromatography KW - design of experiment KW - LC–MS/MS KW - MALDI-TOF-MS Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-559282 SN - 1866-8372 SP - 1 EP - 18 PB - Universitätsverlag Potsdam CY - Potsdam ER - TY - JOUR A1 - Sagu Tchewonpi, Sorel A1 - Huschek, Gerd A1 - Waldbach Braga, Tess A1 - Rackiewicz, Michal A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.) JF - Processes : open access journal N2 - Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples. KW - wheat KW - α-amylase/trypsin inhibitors KW - fractionation KW - purification KW - reversed-phase chromatography KW - ion-exchange chromatography KW - design of experiment KW - LC–MS/MS KW - MALDI-TOF-MS Y1 - 2022 U6 - https://doi.org/10.3390/pr10020259 SN - 2227-9717 VL - 10 SP - 1 EP - 18 PB - MDPI CY - Basel, Schweiz ET - 2 ER - TY - JOUR A1 - Bönick, Josephine A1 - Huschek, Gerd A1 - Rawel, Harshadrai Manilal T1 - Determination of wheat, rye and spelt authenticity in bread by targeted peptide biomarkers JF - Journal of Food Composition and Analysis N2 - Adulteration of food and mislabeled products in global market is a major financial and reputational risk for food manufacturers and trade companies. Consequently, there is a necessity to develop analytical methods to meet these issues. An analytical strategy to check the authenticity of wheat, spelt and rye addition in bread products was developed based on database research, in silico digestion confirming peptide specificity and finally quantification by liquid chromatography-tandem mass spectrometry analysis. Peptide markers for wheat (SQQQISQQPQQLPQQQQIPQQPQQF; QQHQIPQQPQQFPQQQQF and QPHQPQQPYPQQ), spelt (ASIVVGIGGQ; SQQPGQIIPQQPQQPSPL) and rye (LPQSHKQHVGQGAL; AQVQGIIQPQQL and QQFPQQPQQSFPQQPQQPVPQQPL) were identified, verified by protein Basic Local Alignment Search Tool and database research and used for quantification in bread. The specific use of multi-reaction monitoring transitions of selected peptides permitted the identification of closely related species wheat and spelt. Other cereal species (emmer, einkorn, barley, maize, rye and oat) were also checked. The target peptides were quantified at different levels using own reference baked products (bread) after in-solution chymotryptic digestion. Sensitivity of the identification was 0.5-1% using flour-based (0-25%) matrix calibration and the analytical recovery in bread was 80-125%. The analytical strategy described here supplies an emerging, independent and flexible tool in controlling the labeling of bread. KW - Food analysis KW - Food composition KW - Food safety KW - Plant authentication KW - Wheat KW - Spelt KW - Rye KW - LC-MS-MS KW - Quantification of peptides KW - Food labeling Y1 - 2017 U6 - https://doi.org/10.1016/j.jfca.2017.01.019 SN - 0889-1575 SN - 1096-0481 VL - 58 SP - 82 EP - 91 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Huschek, Gerd A1 - Bönick, Josephine A1 - Merkel, Dietrich A1 - Huschek, Doreen A1 - Rawel, Harshadrai Manilal T1 - Authentication of leguminous-based products by targeted biomarkers using high resolution time of flight mass spectrometry JF - LWT - food science and technology : an official journal of the Swiss Society of Food Science and Technology (SGLWT/SOSSTA) and the International Union of Food Science and Technology (IUFoST) N2 - A growing number of health-conscious individuals supplements their diet with protein-rich plant-based products to reduce their meat consumption. Analytical methods are needed to authenticate these new vegetarian products not only for the correct labelling of ingredients according to European legislation but also to discourage food fraud. This paper presents new biomarkers for a targeted proteomics LC-MS/MS work-flow that can simultaneously prove the presence/absence of garden pea, a protein-rich legume, meat and honey and quantify their content in processed vegan food. We show a novel rapid strategy to identify biomarkers for species authentication and the steps for the multi-parameter LC-MS/MS method validation and quantification. A high resolution triple time of flight mass spectrometer (HRMS) with SWATH Acquisition was used for the rapid discovery of all measurable trypsin-digested proteins in the individual ingredients. From these proteins, species-selective biomarkers were identified with BLAST and Skyline. Vicilin and convicilin (UniProt: D3VND9, Q9M3X6) allow pea authentication with regard to other legume species. Myostatin (UniProt: 018831) is a single biomarker for all meat types. For honey, we identified three selective proteins (UniProt: C6K481, C6K482, Q3L6329). The final LC-MS/MS method can identity and quantify these markers simultaneously. Quantification occurs via external matrix calibration. KW - Vegan KW - Food authentication KW - Legume KW - Honey KW - Meat peptide biomarker KW - MS quantification of leguminous additives KW - Food labelling Y1 - 2018 U6 - https://doi.org/10.1016/j.lwt.2017.12.034 SN - 0023-6438 SN - 1096-1127 VL - 90 SP - 164 EP - 171 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Sagu Tchewonpi, Sorel A1 - Huschek, Gerd A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - Effect of sample preparation on the detection and quantification of selected nuts allergenic proteins by LC-MS/MS JF - Molecules : a journal of synthetic chemistry and natural product chemistry / Molecular Diversity Preservation International N2 - 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. KW - nut allergenic proteins KW - protein extraction KW - sample preparation KW - tryptic digestion KW - microwave assisted digestion KW - SDS PAGE KW - LC-MS/MS Y1 - 2021 U6 - https://doi.org/10.3390/molecules26154698 SN - 1420-3049 VL - 26 IS - 15 PB - MDPI CY - Basel ER - TY - GEN A1 - Tchewonpi Sagu, Sorel A1 - Huschek, Gerd A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - Effect of sample preparation on the detection and quantification of selected nuts allergenic proteins by LC-MS/MS T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1157 KW - nut allergenic proteins KW - sample preparation KW - protein extraction KW - tryptic digestion KW - microwave assisted digestion KW - SDS PAGE KW - LC-MS/MS Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-521871 SN - 1866-8372 IS - 15 ER - TY - JOUR A1 - Tchewonpi Sagu, Sorel A1 - Landgräber, Eva A1 - Henkel, Ina M. A1 - Huschek, Gerd A1 - Homann, Thomas A1 - Bußler, Sara A1 - Schlüter, Oliver K. A1 - Rawel, Harshadrai Manilal T1 - Effect of cereal α-amylase/trypsin inhibitors on developmental characteristics and abundance of digestive enzymes of mealworm larvae (Tenebrio molitor L.) JF - Insects N2 - The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant. KW - growth behavior KW - Tenebrio molitor larvae KW - feeding KW - cereal meals KW - α-amylase/trypsin inhibitors KW - digestive enzymes quantification KW - LC-MS/MS Y1 - 2021 U6 - https://doi.org/10.3390/insects12050454 SN - 2075-4450 VL - 12 IS - 5 PB - MDPI CY - Basel ER - TY - GEN A1 - Tchewonpi Sagu, Sorel A1 - Landgräber, Eva A1 - Henkel, Ina M. A1 - Huschek, Gerd A1 - Homann, Thomas A1 - Bußler, Sara A1 - Schlüter, Oliver K. A1 - Rawel, Harshadrai Manilal T1 - Effect of cereal α-amylase/trypsin inhibitors on developmental characteristics and abundance of digestive enzymes of mealworm larvae (Tenebrio molitor L.) T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1153 KW - growth behavior KW - Tenebrio molitor larvae KW - feeding KW - cereal meals KW - α-amylase/trypsin inhibitors KW - digestive enzymes quantification KW - LC-MS/MS Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-520924 SN - 1866-8372 IS - 5 ER - TY - JOUR A1 - Rawel, Harshadrai Manilal A1 - Huschek, Gerd A1 - Sagu Tchewonpi, Sorel A1 - Homann, Thomas T1 - Cocoa Bean Proteins-Characterization, Changes and Modifications due to Ripening and Post-Harvest Processing JF - Nutrients N2 - The protein fractions of cocoa have been implicated influencing both the bioactive potential and sensory properties of cocoa and cocoa products. The objective of the present review is to show the impact of different stages of cultivation and processing with regard to the changes induced in the protein fractions. Special focus has been laid on the major seed storage proteins throughout the different stages of processing. The study starts with classical introduction of the extraction and the characterization methods used, while addressing classification approaches of cocoa proteins evolved during the timeline. The changes in protein composition during ripening and maturation of cocoa seeds, together with the possible modifications during the post-harvest processing (fermentation, drying, and roasting), have been documented. Finally, the bioactive potential arising directly or indirectly from cocoa proteins has been elucidated. The state of the art suggests that exploration of other potentially bioactive components in cocoa needs to be undertaken, while considering the complexity of reaction products occurring during the roasting phase of the post-harvest processing. Finally, the utilization of partially processed cocoa beans (e.g., fermented, conciliatory thermal treatment) can be recommended, providing a large reservoir of bioactive potentials arising from the protein components that could be instrumented in functionalizing foods. KW - cocoa processing KW - cocoa proteins KW - classification KW - extraction and characterization methods KW - fermentation-related enzymes KW - bioactive peptides KW - heath potentials KW - protein-phenol interactions Y1 - 2019 U6 - https://doi.org/10.3390/nu11020428 SN - 2072-6643 VL - 11 IS - 2 PB - MDPI CY - Basel ER -