A concept of dual-responsive prodrugs based on oligomerization-controlled reactivity of ester groups: an improvement of cancer cells versus neutrophils selectivity of camptothecin.

Many known chemotherapeutic anticancer agents exhibit neutropenia as a dose-limiting side effect. In this paper we suggest a prodrug concept solving this problem for camptothecin (HO-cpt). The prodrug is programmed according to Boolean "AND" logic. In the absence of H2O2 (trigger T1), e.g. in the majority of normal cells, it exists as an inactive oligomer. In cancer cells and in primed neutrophils (high H2O2), the oligomer is disrupted forming intermediate (inactive) lipophilic cationic species. These are accumulated in mitochondria (Mit) of cancer cells, where they are activated by hydrolysis at mitochondrial pH 8 (trigger T2) with formation of camptothecin. In contrast, the intermediates remain stable in neutrophils lacking Mit and therefore a source of T2. In this paper we demonstrated a proof-of-concept. Our prodrug exhibits antitumor activity both in vitro and in vivo, but is not toxic to normal cell and neutrophils in contrast to known single trigger prodrugs and the parent drug HO-cpt.

Comprehensive species- and processing-specific peptide profiling of pasteurized, extended shelf-life and ultra-high temperature milk from cow, goat, sheep, buffalo, and mare.

The present study aimed to identify endogenous milk peptides for species differentiation independent of heat exposure. Thus, comprehensive milk peptide profiles from five species and three types of heat treatments were analyzed by micro-flow liquid chromatography ion mobility quadrupole time-of-flight mass spectrometry (microLC-IM-QTOF) with subsequent database search leading to ≥ 3000 identified peptides. In the milks, 1154 peptides were unique for cow, 712 for sheep, 466 for goat, 197 for buffalo, and 69 for mare. Most peptides were detected in extended-shelf life (ESL) milk (2010), followed by ultra-high temperature (UHT) processed (1474) and pasteurized milk (1459 peptides), with 693 peptides present in all milk types. A blind test set of 64 samples confirmed eight species-specific, but heat-independent marker peptides in milk from cow, seven from goat, six from sheep, nine from buffalo, and three from mare. The generated peptide profiles can also be used to identify species- and heat-specific markers.

Identification of Potential Bioactive Peptides in Sheep Milk Kefir through Peptidomic Analysis at Different Fermentation Times.

Sheep farming is an important socioeconomic activity in most Mediterranean countries, particularly Spain, where it contributes added value to rural areas. Sheep milk is used in Spain mainly for making cheese, but it can be used also for making other dairy products, such as the lactic-alcoholic fermentation product known as kefir. Dairy products have health benefits because, among other reasons, they contain molecules with biological activity. In this work, we performed a proteomics strategy to identify the peptidome, i.e., the set of peptides contained in sheep milk kefir fermented for four different periods of time, aiming to understand changes in the pattern of digestion of milk proteins, as well as to identify potential bioactive peptides. In total, we identified 1942 peptides coming from 11 different proteins, and found that the unique peptides differed qualitatively among samples and their numbers increased along the fermentation time. These changes were supported by the increase in ethanol, lactic acid, and D-galactose concentrations, as well as proteolytic activity, as the fermentation progressed. By searching in databases, we found that 78 of the identified peptides, all belonging to caseins, had potential biological activity. Of these, 62 were not previously found in any milk kefir from other animal species. This is the first peptidomic study of sheep milk kefir comprising time-course comparison.

The beer component hordenine inhibits alcohol addiction-associated behaviours in mice.

Alcohol consumption is a widespread behaviour that may eventually result in the development of alcohol use disorder (AUD). Alcohol, however, is rarely consumed in pure form but in fruit- or corn-derived preparations, like beer. These preparations add other compounds to the consumption, which may critically modify alcohol intake and AUD risk. We investigated the effects of hordenine, a barley-derived beer compound on alcohol use-related behaviours. We found that the dopamine D2 receptor agonist hordenine (50 mg/kg) limited ongoing alcohol consumption and prophylactically diminished relapse drinking after withdrawal in mice. Although not having reinforcing effects on its own, hordenine blocked the establishment of alcohol-induced conditioned place preference (CPP). However, it independently enhanced alcohol CPP retrieval. Hordenine had a dose-dependent inhibitory effect on locomotor activity. Chronic hordenine exposure enhanced monoamine tissue levels in many brain regions. Further characterization revealed monoaminergic binding sites of hordenine and found a strong binding on the serotonin and dopamine transporters, and dopamine D3 , and adrenergic α1A and α2A receptor activation but no effects on GABAA receptor or glycinergic signalling. These findings suggest that natural ingredients of beer, like hordenine, may work as an inhibitory and use-regulating factor by their modulation of monoaminergic signalling in the brain.

Identification and quantification of kukoamine A and kukoamine B as novel µ-opioid receptor agonists in potato and other solanaceous plants.

Activation of the µ-opioid receptor (µOR) by food components could lead to reward effects or to the modulation of motor functions in the gastrointestinal tract. In an unbiased search for novel µOR agonists in food, a three-step virtual-screening process selected 22 promising candidates with potential to interact with the µOR. Radioligand binding studies showed that ten of these substances indeed bind to the receptor. Functional assays identified kukoamine A as a full agonist (EC50 = 5.6 µM) and kukoamine B as a partial agonist (EC50 = 8.7 µM) to µOR. After extraction, both kukoamines were analyzed by LC-MS/MS in potato, tomato, pepper, and eggplant. Depending on the potato variety, up to 16 µg of kukoamine A and 157 µg of kukoamine B per gram dry weight could be determined in the whole tuber, mainly concentrated in the potato peel. Cooking did not influence the kukoamine contents.

Identification of colupulone and lupulone as the main contributors to the antibacterial activity of hop extracts using activity-guided fractionation and metabolome analysis.

Hop is widely used in beer brewing and as a medicinal product. The present study comprehensively analyzed the main molecular determinants of the antibacterial activity of hop extracts. Minimum inhibitory concentrations (MIC) against Bacillus subtilis between 31.25 and 250 µg/mL were found in the ethanolic extracts of five hop varieties for beer brewing, but not in the tea hop sample. Activity-guided fractionation revealed the highest antibacterial activity for lupulone and adlupulone (MIC 0.98 µg/mL). Metabolome profiling and subsequent multistep statistical analysis detected 33 metabolites out of 1826 features to be associated with the antibacterial activity including humulone, adhumulone, colupulone, lupulone, and adlupulone. Xanthohumol, the three humulone- and three lupulone congeners were quantified in the hop extracts by a validated ultrahigh-performance liquid chromatography-mass spectrometry method. Considering concentrations and MICs, colupulone and lupulone were identified as major contributors to the antibacterial activity of hop extract with the highest antibacterial activity values (concentration/MIC) of 1.59 and 2.56.

Phosphoproteomic Analysis of Dopamine D2 Receptor Signaling Reveals Interplay of G Protein- and ß-Arrestin-Mediated Effects.

Leveraging biased signaling of G protein-coupled receptors has been proposed as a promising strategy for the development of drugs with higher specificity. However, the consequences of selectively targeting G protein- or ß-arrestin-mediated signaling on cellular functions are not comprehensively understood. In this study, we utilized phosphoproteomics to gain a systematic overview of signaling induced by the four biased and balanced dopamine D2 receptor (D2R) ligands MS308, BM138, quinpirole, and sulpiride in an in vitro D2R transfection model. Quantification of 14,160 phosphosites revealed a low impact of the partial G protein agonist MS308 on cellular protein phosphorylation, as well as surprising similarities between the balanced agonist quinpirole and the inverse agonist sulpiride. Analysis of the temporal profiles of ligand-induced phosphorylation events showed a transient impact of the G protein-selective agonist MS308, whereas the ß-arrestin-preferring agonist BM138 elicited a delayed, but more pronounced response. Functional enrichment analysis of ligand-impacted phosphoproteins and treatment-linked kinases confirmed multiple known functions of D2R signaling while also revealing novel effects, for example of MS308 on sterol regulatory element-binding protein-related gene expression. All raw data were deposited in MassIVE (MSV000089457).

Analysis of Chlorate in Chemical Leavening Agents Used for Bakery Products by Liquid Chromatography-Mass Spectrometry.

Chlorate is a food contaminant that is mainly attributed to the use of chlorinated water and disinfectants. The present study investigated if chlorate could also occur as a process contaminant in chemical leavening agents for baking products. Thus, a sensitive and rapid ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated. Chlorate was quantified using an isotopically labeled internal standard after complete degassing of carbonate-based products. The limit of detection/limit of quantification was 0.02 and 0.1 mg/kg, respectively, with recovery rates between 97.0 and 101.2% (concentration levels: 0.3, 1.4, or 5.0 mg/kg). Samples of baking powder, sodium bicarbonate, ammonium bicarbonate, and potassium carbonate were analyzed. Chlorate was detected in all samples of baking powder in concentrations of 0.23-1.87 mg/kg. Potassium carbonate contained the highest chlorate levels, with a maximum of 60.9 mg/kg. These results indicate that baking powder and, particularly, potassium carbonate can be relevant sources of chlorate in food.

In Vitro Reactivity of the Glucose Degradation Product 3,4-Dideoxyglucosone-3-ene (3,4-DGE) towards Abundant Components of the Human Blood Circulatory System.

3,4-Dideoxyglucosone-3-ene (3,4-DGE) is a glucose degradation product present in processed foods and medicinal products. Additionally, its constant formation from 3-deoxyglucosone in plasma has been suggested. Due to its α,ß-unsaturated dicarbonyl moiety, 3,4-DGE is highly reactive and has shown harmful effects in vitro. Here, we investigated the impact of major components of the human blood circulatory system on 3,4-DGE in vitro. Under physiological conditions, plasma concentrations of human serum albumin (HSA) reacted efficiently with 3,4-DGE, resulting in only 8.5% of the initial 3,4-DGE concentration after seven hours (vs. 83.4% without HSA, p < 0.001). Thereby, accessible thiol groups were reduced from 0.121 to 0.064 mol/mol HSA, whereas ketoprofen binding and esterase-like activity of HSA were not affected. Plasma concentrations of glutathione (GSH) reacted immediately and completely with 3,4-DGE, leading to two stereoisomeric adducts. Plasma concentrations of immunoglobulin G (IgG) bound to 3,4-DGE to a lower extent, resulting in 62.6% 3,4-DGE after seven hours (vs. 82.2% in the control, p < 0.01). Immobilized human collagen type IV did not alter 3,4-DGE concentrations. The results indicated that particularly HSA, GSH, and IgG readily scavenge 3,4-DGE after its appearance in the blood stream, which may be associated with a reduced antioxidative and cytoprotective activity for the living cells and, thus, the human organism by blocking free thiol groups.

Degradation and de novo formation of nine major glucose degradation products during storage of peritoneal dialysis fluids.

Reactive glucose degradation products (GDPs) are formed during heat sterilization of glucose-containing peritoneal dialysis fluids (PDFs) and may induce adverse clinical effects. Long periods of storage and/or transport of PDFs before use may lead to de novo formation or degradation of GDPs. Therefore, the present study quantified the GDP profiles of single- and double-chamber PDFs during storage. Glucosone, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), glyoxal, methylglyoxal (MGO), acetaldehyde, formaldehyde, and 5-hydroxymethylfurfural (5-HMF) were quantified by two validated UHPLC-DAD methods after derivatization with o-phenylenediamine (dicarbonyls) or 2,4-dinitrophenylhydrazine (monocarbonyls). The PDFs were stored at 50 °C for 0, 1, 2, 4, 13, and 26 weeks. The total GDP concentration of single-chamber PDFs did not change considerably during storage (496.6 ± 16.0 µM, 0 weeks; 519.1 ± 13.1 µM, 26 weeks), but individual GDPs were affected differently. 3-DG (- 82.6 µM) and 3-DGal (- 71.3 µM) were degraded, whereas 5-HMF (+ 161.7 µM), glyoxal (+ 32.2 µM), and formaldehyde (+ 12.4 µM) accumulated between 0 and 26 weeks. Acetaldehyde, glucosone, MGO, and 3,4-DGE showed time-dependent formation and degradation. The GDP concentrations in double-chamber fluids were generally lower and differently affected by storage. In conclusion, the changes of GDP concentrations during storage should be considered for the evaluation of clinical effects of PDFs.

Stability and Activity of the Antimicrobial Peptide Leg1 in Solution and on Meat and Its Optimized Generation from Chickpea Storage Protein.

The antimicrobial peptide Leg1 (RIKTVTSFDLPALRFLKL) from chickpea legumin is active against spoilage bacteria, yeast, and mold. The present study tested its effectiveness under food storage conditions and examined options to obtain a food-grade agent. The minimum inhibitory concentration (MIC) of Leg1 against E. coli (62.5 µM) proved stable over seven days at 20 °C or 4 °C. It was not influenced by reduced pH (5.0 vs. 6.8), which is relevant in food such as meat. An incubation temperature of 20 °C vs. 37 °C reduced the MIC to 15.6/7.8 µM against E. coli/B. subtilis. With a minimum bactericidal concentration in meat of 125/15.6 µM against E. coli/B. subtilis, Leg1 is equivalently effective as nisin and 5000-82,000 times more active than sodium benzoate, potassium sorbate, or sodium nitrite. Replacing the counter-ion trifluoroacetate derived from peptide synthesis by the more natural alternatives acetate or chloride did not impair the activity of Leg1. As an alternative to chemical synthesis, an optimized protocol for chymotryptic hydrolysis was developed, increasing the yield from chickpea legumin by a factor of 30 compared to the standard procedure. The present results indicate that food-grade Leg1 could possibly be applicable for food preservation.

Antioxidative, Antifungal and Additive Activity of the Antimicrobial Peptides Leg1 and Leg2 from Chickpea.

The fight against food waste benefits from novel agents inhibiting spoilage. The present study investigated the preservative potential of the antimicrobial peptides Leg1 (RIKTVTSFDLPALRFLKL) and Leg2 (RIKTVTSFDLPALRWLKL) recently identified in chickpea legumin hydrolysates. Checkerboard assays revealed strong additive antimicrobial effects of Leg1/Leg2 with sodium benzoate against Escherichia coli and Bacillus subtilis with fractional inhibitory concentrations of 0.625 and 0.75. Additionally, Leg1/Leg2 displayed antifungal activity with minimum inhibitory concentrations of 500/250 µM against Saccharomyces cerevisiae and 250/125 µM against Zygosaccharomyces bailii. In contrast, no cytotoxic effects were observed against human Caco-2 cells at concentrations below 2000 µM (Leg1) and 1000 µM (Leg2). Particularly Leg2 showed antioxidative activity by radical scavenging and reducing mechanisms (maximally 91.5/86.3% compared to 91.2/94.7% for the control ascorbic acid). The present results demonstrate that Leg1/Leg2 have the potential to be applied as preservatives protecting food and other products against bacterial, fungal and oxidative spoilage.

Functionally selective activation of the dopamine receptor D2 is mirrored by the protein expression profiles.

The development of functionally selective or biased ligands is a promising approach towards drugs with less side effects. Biased ligands for G protein-coupled receptors can selectively induce G protein activation or ß-arrestin recruitment. The consequences of this selective action on cellular functions, however, are not fully understood. Here, we investigated the impact of five biased and balanced dopamine D2 receptor agonists and antagonists on the global protein expression in HEK293T cells by untargeted nanoscale liquid chromatography-tandem mass spectrometry. The proteome analysis detected 5290 protein groups. Hierarchical clustering and principal component analysis based on the expression levels of 1462 differential proteins led to a separation of antagonists and balanced agonist from the control treatment, while the biased ligands demonstrated larger similarities to the control. Functional analysis of affected proteins revealed that the antagonists haloperidol and sulpiride regulated exocytosis and peroxisome function. The balanced agonist quinpirole, but not the functionally selective agonists induced a downregulation of proteins involved in synaptic signaling. The ß-arrestin-preferring agonist BM138, however, regulated several proteins related to neuron function and the dopamine receptor-mediated signaling pathway itself. The G protein-selective partial agonist MS308 influenced rather broad functional terms such as DNA processing and mitochondrial translation.

Generation of antimicrobial peptides Leg1 and Leg2 from chickpea storage protein, active against food spoilage bacteria and foodborne pathogens.

Contamination with bacteria leads to food waste and foodborne diseases with severe consequences for the environment and human health. Aiming to reduce food spoilage and infection, the present study developed novel highly active food-grade antimicrobial peptides affecting a wide range of bacteria. After extraction from chickpea, the storage protein legumin was hydrolyzed by the digestive protease chymotrypsin. Subsequent analysis by ultrahigh-performance micro-liquid chromatography-triple quadrupole time-of-flight tandem mass spectrometry determined the resulting peptide profiles. Virtual screening identified 21 potential antimicrobial peptides in the hydrolysates. Among those, the peptides Leg1 (RIKTVTSFDLPALRFLKL) and Leg2 (RIKTVTSFDLPALRWLKL) exhibited antimicrobial activity against 16 different bacteria, including pathogens, spoilage-causing bacteria and two antibiotic-resistant strains. Leg1/Leg2 showed minimum inhibitory concentrations (MIC) down to 15.6 µmol/L and were thus 10-1,000-fold more active compared to conventional food preservatives. Moreover, Leg1 and Leg2 showed bactericidal activity in contrast to the bacteriostatic activity of conventional preservatives.

Metal cations promote α-dicarbonyl formation in glucose-containing peritoneal dialysis fluids.

Heat sterilization of peritoneal dialysis fluids (PDFs) leads to the formation of glucose degradation products (GDPs), which impair long-term peritoneal dialysis. The current study investigated the effects of metal ions, which occur as trace impurities in the fluids, on the formation of six major α-dicarbonyl GDPs, namely glucosone, glyoxal, methylglyoxal, 3-deoxyglucosone, 3-deoxygalactosone, and 3,4-dideoxyglucosone-3-ene. The chelation of metal ions by 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA) during sterilization significantly decreased the total GDP content (585 µM vs. 672 µM), mainly due to the decrease of the glucose-oxidation products glucosone (14 µM vs. 61 µM) and glyoxal (3 µM vs. 11 µM), but also of methylglyoxal (14 µM vs. 31 µM). The glucose-dehydration products 3-deoxyglucosone, 3-deoxygalactosone, and 3,4-dideoxyglucosone-3-ene were not significantly affected by chelation of metal ions. Additionally, PDFs were spiked with eleven different metal ions, which were detected as traces in commercial PDFs, to investigate their influence on GDP formation during heat sterilization. Iron(II), manganese(II), and chromium(III) had the highest impact increasing the formation of glucosone (1.2-1.5 fold increase) and glyoxal (1.3-1.5 fold increase). Nickel(II) and vanadium(III) further promoted the formation of glyoxal (1.3 fold increase). The increase of the pH value of the PDFs from pH 5.5 to a physiological pH of 7.5 resulted in a decreased formation of total GDPs (672 µM vs 637 µM). These results indicate that the adjustment of metal ions and the pH value may be a strategy to further decrease the content of GDPs in PDFs.

Reactive dicarbonyl compounds cause Calcitonin Gene-Related Peptide release and synergize with inflammatory conditions in mouse skin and peritoneum.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene-Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Influence of the production technology on kefir characteristics: Evaluation of microbiological aspects and profiling of phosphopeptides by LC-ESI-QTOF-MS/MS.

The influence of production technology, namely, temperature, pH and 2-step fermentation (back-slopping approach), on the microbiological characteristics and on the phosphopeptide profile of kefir obtained with kefir grains was investigated. The growth of yeasts, lactic acid bacteria (LAB) and acetic acetic bacteria (AAB) in both grains and kefir was affected by the incubation temperature and by the use of back-slopping. In particular, at 25 °C the microbiota of kefir grains was mainly composed by LAB and yeasts, while at 18 °C yeasts represented the dominant group in kefir. Back-slopping at 25 °C determined a significant increase of AAB. A comprehensive characterization of potentially bioactive peptides, including caseino-phosphopeptides (CPPs), was performed, for the first time, in kefir obtained with kefir grains, using preliminary enrichment on hydroxyapatite followed by dephosphorylation and analysis by Liquid Chromatography-ElectroSpray Ionization-Quadrupole-Time of Flight-tandem mass spectrometry (LC-ESI-QTOF-MS/MS). As a result, seventy-three phosphopeptides, mostly arising from caseins (79% ß-casein, 8% αs1-casein and 9% αs2-casein) and all including from three to five serine residues in their sequences, were identified. Seventy-one of them showed the typical motif "SerP-SerP-SerP-Glu-Glu", which is crucial for the ability of caseins to bind to minerals. Several peptides were observed, for the first time, from the 1-40 region of ß-casein. As for the effect of production technology, phosphopeptide profiles of kefirs obtained at 25 °C and 18 °C were very similar, whereas kefir produced under acidic conditions showed a predominance of smaller peptides, suggesting a higher level of proteolysis. Conversely, kefir obtained through back-slopping at 25 °C contained longer peptides, thus indicating a lower proteolytic activity and a poor reproducibility in the kefir phosphopeptide profile occurring when grains are reused.

Absorption, Biokinetics, and Metabolism of the Dopamine D2 Receptor Agonist Hordenine (N,N-Dimethyltyramine) after Beer Consumption in Humans.

Hordenine, a natural constituent of germinated barley, is a biased agonist of the dopamine D2 receptor. This pilot study investigated the biokinetics of hordenine and its metabolites in four volunteers consuming beer equal to 0.075 mg hordenine/kg body weight. A new ultrahigh-performance liquid chromatography method coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method determined maximum plasma concentrations of 12.0-17.3 nM free hordenine after 0-60 min. Hordenine phase-II metabolism was first dominated by sulfation, but later by glucuronidation. The elimination half-lives in plasma were 52.7-66.4 min for free hordenine and about 60/80 min longer for hordenine sulfate and hordenine glucuronide. Urinary excretion peaked 2-3.5 h after consumption and accumulated to 3.78 µmol within 24 h, corresponding to 9.9% of the ingested dose. The observed hordenine levels in plasma seem too low to provoke direct interaction with the dopamine D2 receptor related to food reward, but synergistic or additive effects with alcohol or N-methyltyramine may occur.

Untargeted Proteomics-Based Profiling for the Identification of Novel Processing-Induced Protein Modifications in Milk.

Nonenzymatic post-translational protein modifications (nePTMs) affect the nutritional, physiological, and technological properties of proteins in food and in vivo. In contrast to the usual targeted analyses, the present study determined nePTMs in processed milk in a truly untargeted proteomic approach. Thus, it was possible to determine to which extent known nePTM structures explain protein modifications in processed milk and to detect and identify novel products. The method combined ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with bioinformatic data analysis by the software XCMS. The nePTMs detected by untargeted profiling of a ß-lactoglobulin-lactose model were incorporated in a sensitive scheduled multiple reaction monitoring method to analyze these modifications in milk samples and to monitor their reaction kinetics during thermal treatment. Additionally, we identified the structures of unknown modifications. Lactosylation, carboxymethylation, formylation of lysine and N-terminus, glycation of arginine, oxidation of methionine, tryptophan, and cysteine, oxidative deamination of N-terminus, and deamidation of asparagine and glutamine were the most important reactions of ß-lactoglobulin during milk processing. The isomerization of aspartic acid was observed for the first time in milk products, and N-terminal 4-imidazolidinone was identified as a novel nePTM.

Identification of peptides reflecting the storage of UHT milk by MALDI-TOF-MS peptide profiling.

Proteolysis during the storage of UHT milk is associated with major technological problems, particularly bitter off-flavors and age gelation limiting the shelf life of milk. In this study, untargeted peptide profiling by MALDI-TOF-MS identified peptides that were formed by proteolysis and reflected the storage of UHT milk. Analysis of nine different commercial UHT samples recorded peptide profiles during and at the end of their shelf life. Relative quantification and sequencing of the peptides revealed that the concentrations of 22 peptides increased significantly during the storage of UHT milk due to the activity of endogenous milk proteases and microbial proteases as well as other unidentified proteolytic mechanisms. Based on highly discriminative AUC values from receiver operator characteristic (ROC) curve analysis, we selected ten peptides as marker candidates. Among those, the peptide ß-casein192-206 (m/z 1668.9) was the most suitable marker differentiating expired-UHT from regular-UHT samples with 100% accuracy. Additionally, ß-casein191-206 (m/z 1782.0) showed 100% specificity and ß-casein139-161 (m/z 2696.4) 100% sensitivity. Thus, ß-casein192-206, either by itself or in combination with ß-casein191-206 and ß-casein139-161, presents a reliable marker to monitor the storage of UHT milk based on proteolytic mechanisms. SIGNIFICANCE: Enzymatic hydrolysis is the main reason why processed milk spoils during storage. The present study recorded peptide profiles to monitor the release or degradation of peptides in stored UHT milk. Among the detected peptides, statistical analysis revealed that the relative concentration of ß-casein192-206 reflected those proteolytic processes most precisely. Food authorities can now refer to ß-casein192-206 as a reliable marker to differentiate between freshly processed milk and products at the end of their shelf life. Furthermore, the food industry can use this marker peptide to improve production processes by monitoring the proteolysis during storage. The recorded peptide profile helps to explain the basic mechanisms leading to storage-induced proteolysis.