TY - JOUR A1 - Sagu Tchewonpi, Sorel A1 - Zimmermann, Lynn A1 - Landgräber, Eva A1 - Homann, Thomas A1 - Huschek, Gerd A1 - Özpinar, Haydar A1 - Schweigert, Florian J. A1 - Rawel, Harshadrai Manilal T1 - Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS JF - Foods N2 - The α-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker’s asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entries in UniProt database. To substantially test proof of concept, 46 German and Turkish wheat samples were used. Two extractions systems based on chloroform/methanol mixture (CM) and under buffered denaturing conditions were evaluated. Three aspects were optimized, tryptic digestion, chromatographic separation, and targeted tandem mass spectrometric analysis (HPLC-MS/MS). Preliminary characterization with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) documented the purity of the extracted ATIs with CM mixture and the amylase (60–80%)/trypsin (10–20%) inhibition demonstrated the bifunctional activity of ATIs. Thirteen (individual/common) biomarkers were established. Major ATIs (7–34%) were differently represented in samples. Finally, to our knowledge, the proposed HPLC-MS/MS method allowed for the first time so far the analysis of all 14 reviewed wheat ATI entries reported. KW - α-amylase/trypsin inhibitors KW - wheat cultivars KW - SDS-PAGE KW - peptides markers KW - relative quantification KW - mass spectrometry KW - LC-MRM-MS Y1 - 2020 U6 - https://doi.org/10.3390/foods9101448 SN - 2304-8158 VL - 9 IS - 10 PB - MDPI CY - Basel ER - TY - GEN A1 - Sagu Tchewonpi, Sorel A1 - Zimmermann, Lynn A1 - Landgräber, Eva A1 - Homann, Thomas A1 - Huschek, Gerd A1 - Özpinar, Haydar A1 - Schweigert, Florian J. A1 - Rawel, Harshadrai Manilal T1 - Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The α-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker’s asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entries in UniProt database. To substantially test proof of concept, 46 German and Turkish wheat samples were used. Two extractions systems based on chloroform/methanol mixture (CM) and under buffered denaturing conditions were evaluated. Three aspects were optimized, tryptic digestion, chromatographic separation, and targeted tandem mass spectrometric analysis (HPLC-MS/MS). Preliminary characterization with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) documented the purity of the extracted ATIs with CM mixture and the amylase (60–80%)/trypsin (10–20%) inhibition demonstrated the bifunctional activity of ATIs. Thirteen (individual/common) biomarkers were established. Major ATIs (7–34%) were differently represented in samples. Finally, to our knowledge, the proposed HPLC-MS/MS method allowed for the first time so far the analysis of all 14 reviewed wheat ATI entries reported. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1028 KW - α-amylase/trypsin inhibitors KW - wheat cultivars KW - SDS-PAGE KW - peptides markers KW - relative quantification KW - mass spectrometry KW - LC-MRM-MS Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-486118 SN - 1866-8372 IS - 1028 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 - TY - JOUR A1 - Rawel, Harshadrai Manilal A1 - Huschek, Gerd A1 - Sagu Tchewonpi, Sorel A1 - Homann, Thomas T1 - Cocoa Bean Proteins BT - 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 - Molecular Diversity Preservation International CY - Basel ER - TY - GEN A1 - Rawel, Harshadrai Manilal A1 - Huschek, Gerd A1 - Sagu Tchewonpi, Sorel A1 - Homann, Thomas T1 - Cocoa Bean Proteins BT - Characterization, Changes and Modifications due to Ripening and Post-Harvest Processing T2 - Postprints der Universität Potsdam: Mathematisch-Naturwissenschaftliche Reihe 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 681 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 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-425953 SN - 1866-8372 IS - 681 ER - TY - GEN A1 - Sagu Tchewonpi, Sorel A1 - Landgräber, Eva A1 - Rackiewicz, Michal A1 - Huschek, Gerd A1 - Rawel, Harshadrai Manilal T1 - Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1068 KW - sorghum KW - α-amylase/trypsin inhibitors KW - reducing agents KW - cysteine alkylation KW - SDS PAGE KW - targeted proteomics KW - LC–MS/MS Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-488096 SN - 1866-8372 IS - 1068 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 - Sagu Tchewonpi, Sorel A1 - Landgräber, Eva A1 - Rackiewicz, Michal A1 - Huschek, Gerd A1 - Rawel, Harshadrai Manilal T1 - Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars JF - Molecules N2 - 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. KW - sorghum KW - α-amylase/trypsin inhibitors KW - reducing agents KW - cysteine alkylation KW - SDS PAGE KW - targeted proteomics KW - LC–MS/MS Y1 - 2020 U6 - https://doi.org/10.3390/molecules25245982 SN - 1420-3049 VL - 25 IS - 24 PB - MDPI CY - Basel ER - TY - JOUR A1 - Figueroa Campos, Gustavo Adolfo A1 - G. K. T. Kruizenga, Johannes A1 - Sagu Tchewonpi, Sorel A1 - Schwarz, Steffen A1 - Homann, Thomas A1 - Taubert, Andreas A1 - Rawel, Harshadrai Manilal T1 - Effect of the post-harvest processing on protein modification in green coffee beans by phenolic compounds JF - Foods : open access journal N2 - The protein fraction, important for coffee cup quality, is modified during post-harvest treatment prior to roasting. Proteins may interact with phenolic compounds, which constitute the major metabolites of coffee, where the processing affects these interactions. This allows the hypothesis that the proteins are denatured and modified via enzymatic and/or redox activation steps. The present study was initiated to encompass changes in the protein fraction. The investigations were limited to major storage protein of green coffee beans. Fourteen Coffea arabica samples from various processing methods and countries were used. Different extraction protocols were compared to maintain the status quo of the protein modification. The extracts contained about 4–8 µg of chlorogenic acid derivatives per mg of extracted protein. High-resolution chromatography with multiple reaction monitoring was used to detect lysine modifications in the coffee protein. Marker peptides were allocated for the storage protein of the coffee beans. Among these, the modified peptides K.FFLANGPQQGGK.E and R.LGGK.T of the α-chain and R.ITTVNSQK.I and K.VFDDEVK.Q of β-chain were detected. Results showed a significant increase (p < 0.05) of modified peptides from wet processed green beans as compared to the dry ones. The present study contributes to a better understanding of the influence of the different processing methods on protein quality and its role in the scope of coffee cup quality and aroma. View Full-Text KW - Arabica coffee KW - coffee processing KW - protein modification KW - bound phenolic compounds KW - peptide biomarkers KW - LC-MS/MS Y1 - 2022 U6 - https://doi.org/10.3390/foods11020159 SN - 2304-8158 VL - 11 PB - MDPI CY - Basel, Schweiz ET - 2 ER -