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An innovative method for the quantification of corticosterone in the urine of C57BL/6J mice by liquid chromatography-electrospray ionization-tandem mass spectrometry was developed. Unconjugated and glucuronidated corticosterone was detected in the urine samples using enzymatic hydrolysis following liquid–liquid extraction. After optimization of the extraction protocol and LC-MS/MS parameters, we performed a validation study using a representative urine pool of C57BL/6J and Naval Medical Research Institute mice. The method shows good linearity (1–5000 fmol/µL) and the calculated limit of quantification amounts to 0.823 fmol/µL. Both intra-day and inter-day variation was ≤10%, while their recoveries amounted to 90.4–110.6% and 99.8%, respectively. Twenty-four hour urine collection of C57BL/6J mice restrained in two different metabolic cage types for two times was used to test the validated method. To control the hydration level of mice, the corticosterone concentration in their urine was normalized to urinary creatinine concentration. Our LC-MS/MS method represents a highly specific analytical tool for the quantification of corticosterone levels in urine samples, assisting in non-invasive monitoring of acute stress levels in laboratory mice.

The growing number of wheat-related allergies worldwide has resulted in a new trend towards gluten-free alternatives. In this context, alternative cereals such as sorghum and oats are attracting new interest. Given the limited data available, the question of whether these cereals are completely safe and gluten-free for allergy sufferers remains open. One of the key steps in protein research is their efficient extraction. In this work, the Osborne sequential extraction method was developed and optimized using the response surface methodology in order to fractionate oat proteins. An optimized desirability of 0.986 was achieved with an extraction time of 4.7 min, a speed of 6, and a sample/solvent ratio of 5. The corresponding optimized responses were 8.7, 4.0, and 5.1% for the extraction yields of the avenin, avenalin, and albumin/globulin fractions, respectively. Further characterization of the extracts was carried out on 24 homogeneous and commercial oat samples via LC-MS/MS, targeting six potentially allergenic proteins. The avenin-E protein featured prominently, with relative contents of 60.7, 32.2, 58.0, and 59.8% in the total extract, avenin, avenalin, and albumin/globulin fractions, respectively, while the Avenin-3, ATI-2, avenin, SSG2, and SSG1 proteins in the total extract showed levels of 16.4, 9.3, 6.6, 4.8, and 2.2%, respectively. The preliminary results of an ELISA performed on the different fractions revealed low levels of gluten (from 1.24 ± 0.14 to 3.61 ± 0.16 mg/kg), which were well below the threshold limit of 20 mg/kg. These results support the hypothesis that oats can be a safe food for people suffering from cereal-related allergies. These results open the door to further studies into the comprehensive characterization of oat proteins.

For many years, coffee has maintained its place in Turkey as Turkish coffee (i.e., boiled, finely ground, unfiltered), and has become globally recognized. However, in recent years, especially for young people, filter coffee has become more popular, instead of Turkish coffee. As is generally known today, climatic conditions and processing techniques have an impact on the constituents of coffee. It is also thought that the finer grinding of Turkish coffee may affect the variability of the coffee contents. The mentioned popularity of the coffee beverage is also causing an increased interest in its nutritional and functional properties as well as its chemical constituents. Therefore, an analytical determination of the coffee ingredients is essential. Taste may be the most important criterion for coffee consumption for most consumers, but the health benefits should not be underestimated. Coffee has an antioxidant effect due to the phenolic compounds present, such as chlorogenic acid. It is also known to prevent cellular damage, which has led to increased interest in its antioxidant properties. The aim of this study was to investigate the total chlorogenic acids (CQAs), caffeine and antioxidant capacities of coffees commonly consumed in Turkey. For this purpose, three green and three roasted coffee samples of the Coffea arabica, Coffea canephora and Coffea liberica varieties were selected for this study and characterized. Different targeted mass spectrometry (LC-MS/MS) methods were developed to encompass the above-mentioned ingredients. The roasting process significantly increased the caffeine content in three roasted samples compared to green coffees. These values ranged from 1.2 g per 100 g dry mass (DM) in green coffees to 2.6 g per 100 g DM in roasted coffees. It could be shown that the most dominant CQA contained was 5-CQA. While the amount of CQA in green coffees is approximately 49–64%, it decreases down to 33–41%, by isomer conversion partly, to 4-CQA and 3-CQA in the roasting process. The highest value was found at 3.5 g/100 g DM. The highest value for the antioxidation capacity was 4.4 g ascorbic acid equivalents/100 g DM roasted coffee and the lowest value was 2.4 g/100 g DM in green coffee. This study was the first attempt to demonstrate the presence of different CQAs, caffeine and antioxidation capacity in coffee beans from the market in Turkey and needs to be followed up further with a larger sample size in future studies.

The post-harvest processing of coffee beans leads to a wide range of reactions involving proteins. The formation of crosslinks between proteins and phenolic compounds present in high concentrations of coffee beans represents one of the most challenging and still not fully characterized reactions. The aim of this work was to assess the presence of products from such reactions in coffee samples, focusing on the adducts between cysteine and chlorogenic acids (CQAs). For this purpose, 19 green and 15 roasted coffee samples of the Coffea arabica, Coffea canephora, and Coffea liberica varieties were selected for this study and basically characterized. Then, targeted liquid chromatography mass spectrometry (LC-MS/MS) methods were developed to assess the formation of adducts between CQA and cysteine, glutathione, and N-acetylcysteine as the amino acid and peptide models, and quantified such adducts in coffee samples. The results of the characterization showed a heterogeneous distribution of the protein content (8.7–14.6%), caffeine (0.57–2.62 g/100 g), and antioxidant capacity (2–4.5 g ascorbic acid/100 g) in Arabica, Canephora, and Liberica samples. Glutamic acid, arginine, and proline were found to be the major amino acids, while 5-CQA (38–76%), 3-CQA (4–13%), and 4-CQA (4–13%) were the most abundant CQA derivatives of all coffee varieties. The model experiments for adduct formation demonstrated that cysteine binds to CQA via thiol groups and 5-CQA initially isomerizes to 3- and 4-CQA, depending on the conditions, allowing cysteine to bind to two different sites on 3-, 4- or 5-CQA molecules, thus, forming six different Cys-CQA adducts with m/z 476. The reaction was more favored at pH 9, and the adducts proved to be stable up to 90 °C for 10 min and up to 28 days at room temperature. The relative quantification of adducts showed peak area values ranging from 1100 to 3000 in green coffee bean samples, while no adducts were detected in roasted coffee beans. Overall, this work was the first attempt to demonstrate the presence of Cys-CQA adducts in coffee beans and paves the way for further investigations of such adduct formation at the protein level.

Food fraud, even when not in the news, is ubiquitous and demands the development of innovative strategies to combat it. A new non-targeted method (NTM) for distinguishing spelt and wheat is described, which aids in food fraud detection and authenticity testing. A highly resolved fingerprint in the form of spectra is obtained for several cultivars of spelt and wheat using liquid chromatography coupled high-resolution mass spectrometry (LC-HRMS). Convolutional neural network (CNN) models are built using a nested cross validation (NCV) approach by appropriately training them using a calibration set comprising duplicate measurements of eleven cultivars of wheat and spelt, each. The results reveal that the CNNs automatically learn patterns and representations to best discriminate tested samples into spelt or wheat. This is further investigated using an external validation set comprising artificially mixed spectra, samples for processed goods (spelt bread and flour), eleven untypical spelt, and six old wheat cultivars. These cultivars were not part of model building. We introduce a metric called the D score to quantitatively evaluate and compare the classification decisions. Our results demonstrate that NTMs based on NCV and CNNs trained using appropriately chosen spectral data can be reliable enough to be used on a wider range of cultivars and their mixes.

κ-casein (κ-CN) is one of the key components in bovine milk, playing a unique role in the structuration of casein micelles. It contains in its chemical structure up to sixteen amino acid residues (mainly serine and threonine) susceptible to modifications, including glycosylation and phosphorylation, which may further be formed during milk processing. In this study, changes in post-translational modification (PTM) of κ-CN during bovine milk fermentation were investigated. One-to-five-day fermented milk samples were produced. A traditional bottom–up proteomics approach was used to establish a multiple-reaction monitoring (MRM) method for relative quantification of κ-CN PTM. Endoproteinase Glu-C was found to efficiently digest the κ-CN molecule. The developed LC-MS method was validated by performing assessments of linearity, precision, repeatability, reproducibility, limit of detection (LOD), and limit of quantification (LOQ). Among the yielded peptides, four of them containing serine and threonine residues were identified and the unmodified as well as the modified variants of each of them were relatively quantified. These peptides were (1) IPTINTIASGEPTSTTE [140, 158], (2) STVATLE [162, 168], (3) DSPE [169, 172], and (4) INTVQVTSTAV [180, 190]. Distribution analysis between unmodified and modified peptides revealed that over 50% of κ-CN was found in one of its modified forms in milk. The fermentation process further significantly altered the composition between unmodified/modified κ-CN, with glycoslaytion being predominant compared to phosphorylation (p < 0.01). Further method development towards α and β-CN fractions and their PTM behavior would be an asset to better understand the changes undergone by milk proteins and the micellar structure during fermentation.

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.

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 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.

Many technical challenges still need to be overcome to improve the quality of the green coffee beans. In this work, the wet Arabica coffee processing in batch and continuous modus were investigated. Coffee beans samples as well as by-products and wastewaters collected at different production steps were analyzed in terms of their content in total phenols, antioxidant capacity, caffeine content, organic acids, reducing sugars, free amino group and protein content. The results showed that 40% of caffeine was removed with pulp. Green coffee beans showed highest concentration of organic acids and sucrose (4.96 ± 0.25 and 5.07 ± 0.39 g/100 g DW for the batch and continuous processing). Batch green coffee beans contained higher amount of phenols. 5-caffeoylquinic Acid (5-CQA) was the main constituent (67.1 and 66.0% for the batch and continuous processing, respectively). Protein content was 15 and 13% in the green coffee bean in batch and continuous processing, respectively. A decrease of 50 to 64% for free amino groups during processing was observed resulting in final amounts of 0.8 to 1.4% in the processed beans. Finally, the batch processing still revealed by-products and wastewater with high nutrient content encouraging a better concept for valorization.

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.

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.