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 - 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 - 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 - JOUR A1 - Reichetzeder, Christoph A1 - Putra, S. E. Dwi A1 - Pfab, T. A1 - Slowinski, T. A1 - Neuber, Corinna A1 - Kleuser, Burkhard A1 - Hocher, Berthold T1 - Increased global placental DNA methylation levels are associated with gestational diabetes JF - Clinical epigenetics N2 - Background: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM. Methods: Global DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study. Results: Mothers with GDM displayed a significantly increased global placental DNA methylation (3.22 +/- 0.63 vs. 3.00 +/- 0.46 %; p = 0.013; +/- SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1-4 (2.9-5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation. Conclusions: This is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors. KW - Placenta KW - Gestational diabetes KW - Insulin resistance KW - LC-MS/MS KW - Global DNA methylation KW - Epigenetics KW - Hypermethylation Y1 - 2016 U6 - https://doi.org/10.1186/s13148-016-0247-9 SN - 1868-7083 VL - 8 PB - BioMed Central CY - London ER - TY - JOUR A1 - Sagu Tchewonpi, Sorel A1 - Huschek, Gerd A1 - Bönick, Josephine A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - A New Approach of Extraction of α-Amylase/trypsin Inhibitors from Wheat (Triticum aestivum L.), Based on Optimization Using Plackett–Burman and Box–Behnken Designs JF - molecules N2 - Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (Triticum aestivum L.), Julius and Ponticus were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (p < 0.01). In both wheat cultivars, Julius and Ponticus, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for Julius and Ponticus, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential. KW - wheat KW - α-amylase/trypsin inhibitors KW - extraction KW - Plackett–Burman design KW - Doehlert design KW - SDS-PAGE KW - MALDI-TOF/MS KW - LC-MS/MS Y1 - 2019 U6 - https://doi.org/10.3390/molecules24193589 SN - 1420-3049 VL - 24 IS - 19 PB - MDPI CY - Basel ER - TY - JOUR A1 - Putra, Sulistyo Emantoko Dwi A1 - Neuber, Corinna A1 - Reichetzeder, Christoph A1 - Hocher, Berthold A1 - Kleuser, Burkhard T1 - Analysis of genomic DNA methylation levels in human placenta using liquid Chromatography-Electrospray ionization tandem mass spectrometry JF - Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry and pharmacology N2 - Background: DNA-methylation is a common epigenetic tool which plays a crucial role in gene regulation and is essential for cell differentiation and embryonic development. The placenta is an important organ where gene activity can be regulated by epigenetic DNA modifications, including DNA methylation. This is of interest as, the placenta is the interface between the fetus and its environment, the mother. Exposure to environmental toxins and nutrition during pregnancy may alter DNA methylation of the placenta and subsequently placental function and as a result the phenotype of the offspring. The aim of this study was to develop a reliable method to quantify DNA methylation in large clinical studies. This will be a tool to analyze the degree of DNA methylation in the human placenta in relationship to clinical readouts. Methods: Liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS) technique was used for the quantification of the 5dmC/dG ratio in placentas from 248 healthy pregnancies. We were able to demonstrate that this method is a reliable and stable way to determine global placental DNA methylation in large clinical trials. Results/Conclusion: The degree of placental DNA methylation seen in our pilot study varies substantially from 2% to 5%. The clinical implications of this variation need to be demonstrated in adequately powered large studies. KW - Pregnancy KW - Placenta KW - Methylation KW - Global KW - LC-MS/MS KW - Fetal programming KW - Clinical Y1 - 2014 U6 - https://doi.org/10.1159/000358666 SN - 1015-8987 SN - 1421-9778 VL - 33 IS - 4 SP - 945 EP - 952 PB - Karger CY - Basel ER -