In this study, a new approach was introduced to identify marker peptides that reflect the thermal treatment of commercial milk samples and differentiate ultrahigh-temperature processed (UHT) milk from mildly heated milk. Peptide profiles of training set samples, pasteurized (n = 20), extended shelf life (n = 29), and UHT (n = 29) milk, were recorded by MALDI-TOF-MS after StageTip microextraction. As marker candidates, 13 peptides were selected, and their cutoff levels were defined. The quality of the cutoff levels was then tested with a blind test set. Thus, the peptide m/z 1701.0, which was identified as pyroQ-βcasein194–209, could ideally differentiate UHT milk from mildly heated milk with an accuracy of 100%. Due to its high reliability and sensitivity, this peptide may be applied in routine analysis to monitor thermal processing of milk. An additional heating experiment showed that the marker peptide candidates are formed during milk processing by endogenous enzymes and selective thermal cleavage.Keywords: heating; MALDI-TOF-MS; marker peptide; milk; PCA; peptide profiling;

•Activation of human α1β2-GABAA receptor by spice extracts was investigated in vitro.•GABAA receptors are an emerging drug target for analgesic effects.•Aqueous clove bud extracts showed highest potentiation of GABA activity.•Eugenol was the main contributor of the GABAergic activity of the clove extract.•Acetyleugenol was even more active than eugenol, but only present in trace amounts.The spice Syzygium aromaticum L. (clove buds) exerts topical anesthetic and analgesic effects. Since GABAA receptors are an emerging drug target for pain treatment, the effects of aqueous clove extracts on the human α1β2-GABAA receptor were tested by two-electrode voltage clamp technique applying a three-step test system. The extract significantly and specifically potentiated the GABA-induced currents by an allosteric mechanism in concentration-dependent manner (0.5–5 µg/mL; up to 426 ± 23%). HPLC-based activity-guided fractionation revealed eugenol as main determinant of this GABAergic activity. Acetyleugenol, an important component of clove bud oil, showed even higher activity than eugenol (1 µg/mL; 308 ± 26% versus 234 ± 29%), but was detected in the aqueous extract only in trace amounts. Thus, the analgesic effects of clove might be partially mediated by positive modulation of the GABAA receptor and eugenol is a major contributor to this activity.Download high-res image (77KB)Download full-size image

•Selected reaction monitoring suited for cytoprotective enzyme expression analysis•Isotope-coded protein labels allow for analysis of primary human colon cells•Primary human cells are suited to monitor nutritive protection against colon cancer.•Cinnamaldehyde and carnosol were the most potent inducers of cytoprotective enzymes.•NAD(P)H dehydrogenase [quinone] 1 was most susceptible to nutritive modulation.Cytoprotective effects by upregulating cellular expression levels of antioxidant proteins are attributed to a significant number of food ingredients. Evaluation of those cytoprotective effects and identification of the most active components requires reliable and comprehensive proteomic strategies. Thus, promising potential bioactive ingredients could be determined for the prevention of various diseases, including colon cancer formation. We established a multiplexed nanoLC–MS/MS targeted proteomic method, operated in scheduled selected reaction monitoring mode (sSRM), to record modulation of the expression levels of six major antioxidant proteins induced by dietary phytochemicals. Relative quantification was achieved by isotope-coded protein labels (ICPLs) and based on two to three proteotypic peptides per target protein. The assay provided accurate (mean relative error 6.4%) and precise (mean RSD 7.4%) quantification. Incubation experiments were carried out in primary human colon epithelial cells (HCoEpiCs) and revealed significant upregulation of NAD(P)H dehydrogenase [quinone] 1 (up to threefold) and thioredoxin reductase 1 (up to twofold) by 10 μM sulforaphane (from broccoli), 5 μM carnosol (rosemary), and 20 μM cinnamaldehyde (cinnamon). The latter two substances additionally upregulated heme oxygenase-1 and were identified as the most active components in the test set. The results provide additional evidence for nutritive cytoprotection in human colon cells.SignificanceTargeted proteome analysis using LC coupled to scheduled selected reaction monitoring (sSRM)-MS is a highly flexible and reliable method to monitor protein expression profiles. The present study screened modulators occurring in food, which may be protective against colon cancer by inducing cytoprotective enzymes. Primary human colonic epithelial cells were used because they model the conditions in healthy gut tissue better than immortalized cells. Thus, an LC–MS/MS-sSRM protocol was established and validated including relative quantification of cytoprotective protein expression by isotope-coded protein labels, because metabolic labelling cannot be applied for primary cells. The present study demonstrated that the major components of cinnamon and rosemary, respectively, i.e. cinnamaldehyde and carnosol, are potent alimentary candidates to increase the expression of cytoprotective enzymes in the human colon. Among the investigated enzymes, NAD(P)H dehydrogenase [quinone] 1 (NQO1) was most susceptible towards modulation by phytochemicals. NQO1 exerts its cytoprotective activity by detoxifying electrophilic and oxidative xenobiotics with quinone structure.Download high-res image (289KB)Download full-size image

Health-promoting effects of kefir may be partially caused by bioactive peptides. To evaluate their formation or degradation during gastrointestinal digestion, we monitored changes of the peptide profile in a model of (1) oral, (2) gastric, and (3) small intestinal digestion of kefir. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses revealed clearly different profiles between digests 2/3 and kefir/digest 1. Subsequent ultraperformance liquid chromatography–electrospray ionization–tandem mass spectrometry identified 92 peptides in total (25, 25, 43, and 30, partly overlapping in kefir and digests 1, 2, and 3, respectively), including 16 peptides with ascribed bioactivity. Relative quantification in scheduled multiple reaction monitoring mode showed that many bioactive peptides were released by simulated digestion. Most prominently, the concentration of angiotensin-converting enzyme inhibitor β-casein203–209 increased approximately 10 000-fold after combined oral, gastric, and intestinal digestion. Thus, physiological digestive processes may promote bioactive peptide formation from proteins and oligopeptides in kefir. Furthermore, bioactive peptides present in certain compartments of the gastrointestinal tract may exert local physiological effects.Keywords: bioactive peptides; gastrointestinal digestion; kefir; peptide profile;

•Cytoprotection of neuroblastoma cells by pomegranate wine extract was studied.•Extracts significantly activated Nrf2 and inhibited NF-κB translocation.•Extracts also activated heme oxygenase expression and superoxide dismutase activity.•The effects are mediated by the phenols gallic acid, ellagic acid and punicalagin.Pomegranate (Punica granatum L.) phenolic compounds may prevent oxidative stress-induced neurodegenerative diseases. The present study investigated cytoprotective effects of pomegranate wine extracts and their phenolic compounds in neuroblastoma cells SH-SY5Y. The extracts significantly activated nuclear factor erythroid 2-related factor 2 (Nrf2; ≤2.6-fold) and inhibited nuclear factor κB (NF-κB) nuclear translocation (≥26% activity) without cytotoxic effects. Moreover, pomegranate wine extract time-dependently elevated heme oxygenase-1 (HO-1) expression (≤2.9-fold) and promoted superoxide dismutase (SOD) activity (≤1.4-fold), while it had no significant effect on glutathione peroxidase (GPx) activity. The main phenolic components of the extracts, gallic acid, ellagic acid, and punicalagin also led to a significant inhibition of NF-κB and the activation of Nrf2 translocation and subsequent increase of HO-1 expression and SOD activity, dependent on concentration and incubation time. Thus, pomegranate wine and its phenolic components may have a protective effect on neuronal cells by triggering Nrf2 and inhibiting NF-κB signaling.

•S-allyl-l-cysteine and isoliquiritigenin improved mitochondrial membrane potential.•Isoliquiritigenin enhanced the cell viability in a cell model of nitrosative stress.•Isoliquiritigenin increased the ATP level in a cell model of nitrosative stress.Oxidative and nitrosative stress resulting in mitochondrial dysfunction are an early event in the pathogenesis of Alzheimer’s disease (AD). Nuclear factor erythroid-2-related factor 2 (Nrf2) is a key transcription factor and regulator of the cellular response to oxidative stress. Thus known Nrf2 activators from food materials were tested for improvement of mitochondrial membrane potential (MMP) and ATP level in neuronal pheochromocytoma cell (PC12) models of oxidative and nitrosative stress. The effects of rotenone and sodium nitroprusside (complex inhibitors of the respiratory chain) on mitochondrial function were also studied. Furthermore, Nrf2 activators were tested in human embryonic kidney cells bearing the Swedish mutation of amyloid precursor protein (APPsw HEK cells) as a cellular model of familial AD. Preincubation with S-allyl-l-cysteine and isoliquiritigenin increased MMP in both PC12 cell models in a similar range as the positive control l-sulforaphane. None of the test compounds, however, improved MMP and ATP level in APPsw HEK cells.

•Peptide profiling of pasteurized, ESL and UHT milk was performed by MALDI–TOF–MS.•The intensity of sixteen peptides was dependent on processing conditions.•Heating and storage experiments suggested mechanism for peptide modulation.•The peptide β-casein 196–209 was most heavily influenced by heat treatment.Peptide profiles of different drinking milk samples were examined to study how the peptide fingerprint of milk reflects processing conditions. The combination of a simple and fast method for peptide extraction using stage tips and MALDI–TOF–MS enabled the fast and easy generation and relative quantification of peptide fingerprints for high-temperature short-time (HTST), extended shelf life (ESL) and ultra-high temperature (UHT) milk of the same dairies. The relative quantity of 16 peptides changed as a function of increasing heat load. Additional heating experiments showed that among those, the intensity of peptide β-casein 196–209 (m/z 1460.9 Da) was most heavily influenced by heat treatment indicating a putative marker peptide for milk processing conditions. Storage experiments with HTST- and UHT milk revealed that the differences between different types of milk samples were not only caused by the heating process. Relevant was also the proteolytic activity of enzymes during storage, which were differently influenced by the heat treatment. These results indicate that the peptide profile may be suitable to monitor processing as well as storage conditions of milk.SignificanceIn the present study, peptide profiling of different types of milk was carried out by MALDI–TOF–MS after stage-tip extraction and relative quantification using an internal reference peptide. Although MALDI–TOF–MS covers only part of the peptidome, the method is easy and quick and is, therefore, suited for routine analysis to address several aspects of food authenticity. Using this method, 16 native peptides were detected in milk that could be modulated by different industrial processes. Subsequent heating and storage experiments with pasteurized and UHT milk confirmed that these peptides are indeed related to the production or storage conditions of the respective products. Furthermore, the heating experiments revealed one peptide, namely the β-casein-derived sequence β-casein 196–209, which underwent particularly sensitive modulation by heat treatment. The present results indicate that the modulated peptides, and especially β-casein 196–209, may be suitable markers to monitor processing parameters for industrial milk production. Furthermore, the model experiments suggest mechanisms leading to the formation or degradation of peptides, which help to evaluate putative marker peptides.

Thermal treatment of milk and milk products leads to protein oxidation, mainly the formation of methionine sulfoxide. Reactive oxygen species, responsible for the oxidation, can be generated by Maillard reaction, autoxidation of sugars, or lipid peroxidation. The present study investigated the influence of milk fat on methionine oxidation in milk. For this purpose, quantitative methionine sulfoxide profiling of all ten methionine residues of β-lactoglobulin, α-lactalbumin, and αs1-casein was carried out by ultrahigh-performance liquid chromatography–electrospray ionization tandem mass spectrometry with scheduled multiple reaction monitoring (UHPLC–ESI–MS/MS–sMRM). Analysis of defatted and regular raw milk samples after heating for up to 8 min at 120 °C and analysis of ultrahigh-temperature milk samples with 0.1%, 1.5%, and 3.5% fat revealed that methionine oxidation of the five residues of the whey proteins and of residues M 123, M 135, and M 196 of αs1-casein was not affected or even suppressed in the presence of milk fat. Only the oxidation of residues M 54 and M 60 of αs1-casein was promoted by lipids. In evaporated milk samples, formation of methionine sulfoxide was hardly influenced by the fat content of the samples. Thus, it can be concluded that lipid oxidation products are not the major cause of methionine oxidation in milk.

•Development and validation of a UHPLC-DAD-MS/MS method for pGDP analysis.•Quantification of pGDPs in icodextrin-based peritoneal dialysis fluids.•4-Deoxyglucosone is the main degradation product with concentrations up to 20 μM.•In contrast to glucose-based products, 3-deoxygalactosone is predominant over 3-DG.•Total pGDP contents in the commercial products were between 36.5 and 43.0 μM.During heat sterilization of peritoneal dialysis (PD) fluids, the glucose component is partially degraded. The formed glucose degradation products impair biocompatibility and limit the long-term application of PD fluids. As an alternative to glucose, icodextrin, a polyglucose, is used as osmotic agent in PD fluids. After targeted screening for reactive carbonyl compounds, NMR- and MS-analyses very recently revealed 4-deoxyglucosone (4-DG), 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxypentosone (3,4-DDPS), and 5-hydroxymethylfurfural (5-HMF) as main polyglucose degradation products (pGDPs) in icodextrin-based PD fluids. Now, the present study established and validated a UHPLC method with DAD as well as a UHPLC-MS/MS method for the first-time quantification of those five major pGDPs in commercial icodextrin PD fluids after derivatization with o-phenylenediamine. Thus, 4-DG was identified to be the main degradation product (in concentrations up to 20 μM). In contrast to the values measured in glucose-based products, the concentration of 3-DGal (≤16 μM) was higher than the concentration of 3-DG (≤7 μM) indicating different reaction pathways starting from polyglucose compared to glucose. The compounds 3,4-DDPS and 5-HMF were present in minor quantities (≤0.3 μM each).

Potentiation of γ-amino butyric acid (GABA)-induced GABAA receptor (GABAAR) activation is a common pathway to achieve sedative, sleep-enhancing, anxiolytic, and antidepressant effects. Presently, a three-component test system was established for the identification of novel GABAAR modulating food plants. In the first step, potentiation of GABA-induced response of the GABAAR was analysed by two-electrode voltage clamp (TEVC) for activity on human α1β2-GABAAR expressed in Xenopus laevis oocytes. Positively tested food plants were then subjected to quantification of GABA content by high-performance liquid chromatography with fluorescence detection (HPLC–FLD) to exclude test foods, which evoke a TEVC-response by endogenous GABA. In the third step, specificity of GABAA-modulating activity was assessed by TEVC analysis of Xenopus laevis oocytes expressing the homologous glycine receptor (GlyR). The three-component test was then applied to screen 10 aqueous extracts of food plants for their GABAAR activity. Thus, hop cones (Humulus lupulus) and Sideritis sipylea were identified as the most potent specific GABAAR modulators eliciting significant potentiation of the current by 182 ± 27 and 172 ± 19 %, respectively, at the lowest concentration of 0.5 μg/mL. The extracts can now be further evaluated by in vivo studies and by structural evaluation of the active components.

Heat sterilization of peritoneal dialysis (PD) fluids leads to partial degradation of the osmotic agent to form reactive carbonyl structures, which significantly reduce the biocompatibility of PD fluids and impair long-term PD therapy. Hence, it is important to know the exact composition of the degradation products to improve biocompatibility of PD fluids. Our study conducted targeted screening for degradation products in polyglucose (icodextrin)-containing PD fluids (pGDPs) by applying o-phenylenediamine (OPD) to form stable derivatives, which were analyzed by ultrahigh-performance liquid chromatography with hyphenated diode array tandem mass spectrometry (UHPLC–DAD–MS/MS). For the first time, specific degradation products of polyglucose, namely, 4-deoxyglucosone (4-DG) and 3,4-dideoxypentosone (3,4-DDPS), could be identified in PD fluids. Further, a reaction product of 5-hydroxymethylfurfural (5-HMF) and OPD could be characterized to be (5-(1H-benzo[d]imidazol-2-yl)furan-2-yl)methanol. Additionally, 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal), both known to be present in glucose-based PD fluids, were also detected in polyglucose-containing fluids. Trapping a hitherto unknown degradation product with OPD yielded 1,4-bis(1H-benzo[d]imidazol-2-yl)-3,4-dihydroxybutan-1-one, which was present in heat- as well as filter-sterilized PD fluids.

•Comprehensive profiling identified 257 peptides in kefir.•Among those, 236 were unique to the product.•16 bioactive, e.g. ACE-inhibitory or antimicrobial peptides were revealed.•Most peptides were released from caseins during fermentation by the microflora.•Composition and quantity of peptides can be controlled by the chosen starter culture.Kefir has a long tradition in human nutrition due to its presupposed health promoting effects. To investigate the potential contribution of bioactive peptides to the physiological effects of kefir, comprehensive analysis of the peptide profile was performed by nano-ESI-LTQ-Orbitrap MS coupled to nano-ultrahigh-performance liquid chromatography. Thus, 257 peptides were identified, mainly released from β-casein, followed by αS1-, κ-, and αS2-casein. Most (236) peptides were uniquely detected in kefir, but not in raw milk indicating that the fermentation step does not only increase the proteolytic activity 1.7- to 2.4-fold compared to unfermented milk, but also alters the composition of the peptide fraction. The influence of the microflora was determined by analyzing kefir produced from traditional kefir grains or commercial starter culture. Kefir from starter culture featured 230 peptide sequences and showed a significantly, 1.4-fold higher proteolytic activity than kefir from kefir grains with 127 peptides. A match of 97 peptides in both varieties indicates the presence of a typical kefir peptide profile that is not influenced by the individual composition of the microflora. Sixteen of the newly identified peptides were previously described as bioactive, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, immunomodulating, opioid, mineral binding, antioxidant, and antithrombotic effects.Biological significanceThe present study describes a comprehensive peptide profile of kefir comprising 257 sequences. The peptide list was used to identify 16 bioactive peptides with ACE-inhibitory, antioxidant, antithrombotic, mineral binding, antimicrobial, immunomodulating and opioid activity in kefir. Furthermore, it was shown that a majority of the kefir peptides were not endogenously present in the raw material milk, but were released from milk caseins by proteases of the microbiota and are therefore specific for the product. Consequently, the proteolytic activity and the composition of the peptide profile can be controlled by the applied microflora (grains or starter culture). On the other hand, a considerable portion of the peptide profile was identified to be typical for kefir in general and independent from production parameters.In summary, the generated kefir peptide profile helped to reveal its origin and to identify bioactive peptides in kefir, which may advance the understanding of health benefits of this food product. The results further indicate that subsets of the kefir peptide list can be used as markers to control food authenticity, for example, to distinguish different types of kefir.

•Comprehensive physiological assessment of food demands recording of activity profiles.•Newly developed LC/SRM method reliably monitors enzyme expression profiles.•Coffee increases the expression of heme oxygenase-1, but not of catalase.•Targeted proteome analysis methods have the potential for multiplexed assays.Comprehensive physiological food assessment requires recording of activity profiles. To elucidate the nutritive regulation of antioxidant enzymes, a generally applicable targeted MS method was established for the expression analysis of catalase and then adapted to heme oxygenase-1. Before tryptic digestion, target proteins were prefractionated by off-gel IEF of stimulated and control cell lysate. Targeted proteome analysis was achieved by LC coupled with scheduled selected reaction monitoring MS using 2 proteotypic peptides per protein and 3–4 transitions per peptide. Relative quantification was performed by stable isotope labeling by amino acids in cell culture (SILAC). The assay showed good correlation with results by Western blot. Linearity, precision, and sensitivity were even improved (LC/SRM vs. Western blot: 3 vs. 1 orders of magnitude, RSD 3.7–13.7% vs. 18.4%, LOD 0.2 vs. 1.6 μg/mL). The developed method indicated that coffee does not modulate catalase expression in macrophages (T7cat 103 ± 22%, T17cat 103 ± 16%, p > 0.05 vs. control), but leads to a highly significant increase of heme oxygenase-1 expression (T15Ho-1 420 ± 24%, T22Ho-1 364 ± 37%, p < 0.001 vs. control, p > 0.05 T15Ho-1 vs. T22Ho-1). In regard to multiplex options of the method, targeted proteome analysis can be a valuable tool for the comprehensive analysis of cellular effects of food components.Biological significanceIn the present study, targeted mass spectrometry was applied to determine the influence of food components on the expression of antioxidative enzymes. The results implicate that targeted proteomics may develop into a valuable tool in food science and nutrition to determine the physiological effects of nutrients. In contrast to conventional methods for expression analysis, targeted proteome analysis can be applied to monitor the effects of a food component on a broad range of cellular targets in parallel. Additionally, proteins or protein modifications can be addressed which elude immunochemical methods.

•H2O2-producing Maillard reaction products were extracted by 85% ethanol (MRM85EtOH).•MRM85EtOH inhibited bacterial growth of Escherichia coli.•MRM85EtOH incorporated into packaging material continuously released H2O2.•Packaging material significantly reduced bacterial growth by 5 log-cycles.Active packaging foils with incorporated antimicrobial agents release the active ingredient during food storage. Maillard reaction products (MRPs) show antimicrobial activity that is at least partially mediated by H2O2. De novo generation of H2O2 by an MRP fraction, extracted from a ribose/lysine Maillard reaction mixture by 85% ethanol, was monitored at three concentrations (1.6, 16.1, and 32.3 g/L) and three temperatures (4, 25, and 37 °C) between 0 and 96 h, reaching a maximum of 335 μM H2O2 (32.3 g/L, 37 °C, 96 h). The active MRP fraction (16.1 g/L) completely inhibited the growth of Escherichia coli for 24 h and was therefore incorporated in a polyvinyl acetate-based lacquer and dispersed onto a low-density polyethylene film. The coated film generated about 100 μM H2O2 and resulted in a log-reduction of >5 log-cycles against E. coli. Thus, MRPs can be considered as active ingredients for antimicrobial packaging materials.

In peritoneal dialysis (PD), glucose degradation products (GDPs), which are formed during heat sterilization of dialysis fluids, lead to structural and functional changes in the peritoneal membrane, which eventually result in the loss of its ultrafiltration capacity. To determine the molecular mechanisms behind these processes, the present study tested the influence of the six major α-dicarbonyl GDPs in PD fluids, namely, glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and glucosone with respect to their potential to impair the enzymatic activity of RNase A as well as their effects on cell viability. For comprehensive risk assessment, the α-dicarbonyl GDPs were applied separately and in concentrations as present in conventional PD fluids. Thus, it was shown that after 5 days, glucosone impaired RNase A activity most distinctly (58% remaining activity, p < 0.001 compared to that of the control), followed by 3,4-DGE (62%, p < 0.001), 3-DGal (66%, p < 0.001), and 3-DG (76%, p < 0.01). Methylglyoxal and glyoxal caused weaker inactivation with significant effects only after 10 days of incubation (79%, 81%, p < 0.001). Profiling of the advanced glycation end products formed during the incubation of RNase A with methylglyoxal revealed predominant formation of the arginine modifications imidazolinone, CEA/dihydroxyimidazoline, and tetrahydropyrimidine at Arg10, Arg33, Arg39, and Arg85. Particularly, modification at Arg39 may severely affect the active site of the enzyme. Additionally, structure- and concentration-specific assessment of the cytotoxicity of the α-dicarbonyl GDPs was performed. Although present at very low concentration, the cytotoxic effect of PD fluids after 2 days of incubation was exclusively caused by 3,4-DGE (14% cell viability, p < 0.001). After 4 days of incubation, 3-DGal (13% cell viability, p < 0.001), 3-DG (24%, p < 0.001), and, to a lower extent, glyoxal and methylglyoxal (both 57%, p < 0.01) also reduced cell viability significantly. In conclusion, 3,4-DGE, 3-DGal, and glucosone appear to be the most relevant parameters for the biocompatibility of PD fluids.

Site-specific relative quantification of β-lactoglobulin modifications in heated milk and dairy products was performed to determine their thermal and nonthermal origins and to evaluate marker candidates for milk processing. Therefore, formation kinetics of 19 different structures at 26 binding sites were analyzed by ultrahigh-performance liquid chromatography–tandem mass spectrometry with multiple reaction monitoring (UHPLC-MS/MS/MRM) after specific protein hydrolysis. The results indicate that (i) site-specific analysis of lactulosyllysine may be a more sensitive marker for mild heat treatment than its overall content; (ii) Nε-carboxymethyllysine, N-terminal ketoamide, and asparagine deamidation are of thermal origin and may be good markers for rather intensive heat treatment, whereas Nε-carboxyethyllysine reflects thermal and nonthermal processes; (iii) the relevance of methylglyoxal-derived arginine modifications is low compared to that of other modifications; (iv) oxidation of methionine and cysteine is a rather weak indicator of thermal impact; and (v) the tryptophan modifications formylkynurenine and kynurenine are of nonthermal origin and further degraded during processing.

Milk is an excellent source of bioactive peptides. However, the composition of the native milk peptidome has only been partially elucidated. The present study applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) directly or after prefractionation of the milk peptides by reverse-phase high-performance liquid chromatography (RP-HPLC) or OFFGEL fractionation for the comprehensive analysis of the peptide profile of raw milk. The peptide sequences were determined by MALDI-TOF/TOF or nano-ultra-performance liquid chromatography–nanoelectrospray ionization-LTQ-Orbitrap-MS. Direct MALDI-TOF-MS analysis led to the assignment of 57 peptides. Prefractionation by both complementary methods led to the assignment of another 191 peptides. Most peptides originate from αS1-casein, followed by β-casein, and αS2-casein. κ-Casein and whey proteins seem to play only a minor role as peptide precursors. The formation of many, but not all, peptides could be explained by the activity of the endogenous peptidases, plasmin or cathepsin D, B, and G. Database searches revealed the presence of 22 peptides with established physiological function, including those with angiotensin-converting-enzyme (ACE) inhibitory, immunomodulating, or antimicrobial activity.

Nonenzymatic post-translational protein modifications (nePTMs) result in changes of the protein structure that may severely influence physiological and technological protein functions. In the present study, ultrahigh-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) was applied for the systematic identification and site-specific analysis of nePTMs of β-lactoglobulin in processed milk. For this purpose, β-lactoglobulin, which had been heated with lactose under conditions to force nePTM formation (7 d/60 °C), was screened for predicted modifications by using full scans and enhanced resolution scan experiments combined with enhanced product ion scans. Thus, the main glycation, glycoxidation, oxidation, and deamidation products of lysine, arginine, methionine, cysteine, tryptophan, and asparagine, as well as the N-terminus, were identified. Using these MS data, a very sensitive scheduled multiple reaction monitoring method suitable for the analysis of milk products was developed. Consequently, 14 different PTM structures on 25 binding sites of β-lactoglobulin were detected in different milk products.

Multiphosphorylated peptides endogenously present in milk exert anticariogenic activity due to their calcium binding capacity. This study performed comprehensive analysis of multiphosphorylated peptides in raw milk using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Since phosphopeptides are often negatively discriminated during ionization, putative phosphopeptides were identified by three different methods: (i) selective detection in 4-chloro-α-cyanocinnamic acid MALDI matrix compared to α-cyano-4-hydroxycinnamic acid; (ii) higher relative signal intensity in negative compared to positive ionization mode; and (iii) detection of signal pairs with mass differences of −80 Da or multiples thereof before and after enzymatic dephosphorylation. Thus, 18 putative phosphopeptides from raw milk were annotated. Peptide structures were then determined by product ion spectra from targeted liquid chromatography electrospray ionization tandem-MS analysis. Thus, β-casein33–48, β-casein29–48, β-casein1–21, β-casein2–25, β-casein1–25, β-casein1–27, β-casein1–28, β-casein1–29, β-casein1–32, αS2-casein1–21, and αS2-casein1–24 were revealed as major peptides with one or four phosphorylation sites in raw milk.

Sugar-sweetened carbonated soft drinks (CSDs) are broadly consumed worldwide. The added sugar, particularly high-fructose corn syrup (HFCS), can be an important source of sugar degradation products, such as α-dicarbonyl compounds. This study recorded the α-dicarbonyl profile in CSDs by ultrahigh-performance liquid chromatography with hyphenated diode array–tandem mass spectrometry after derivatization with o-phenylenediamine. Thus, 3-deoxy-d-erythro-hexos-2-ulose (3-DG), d-lyxo-hexos-2-ulose (glucosone), 3-deoxy-d-threo-hexos-2-ulose (3-DGal), 1-deoxy-d-erythro-hexos-2,3-diulose (1-DG), 3,4-dideoxyglucosone-3-ene (3,4-DGE), methylglyoxal, and glyoxal were identified as major α-dicarbonyls and, with the exception of glyoxal, quantified (recovery rates, 85.6–103.1%; RSD, 0.8–3.6%). Total α-dicarbonyl concentration in 25 tested commercial products ranged between 0.3 and 116 μg/mL and was significantly higher in HFCS-sweetened CSDs compared to CSDs sweetened with HFCS and sucrose or with sucrose alone. Predominant was 3-DG (≤87 μg/mL) followed by glucosone (≤21 μg/mL), 3-DGal (≤7.7 μg/mL), 1-DG (≤2.8 μg/mL), methylglyoxal (≤0.62 μg/mL), and 3,4-DGE (≤0.45 μg/mL).

•Carbohydrate degradation products can be formed by heat sterilization of drugs.•They are clinically relevant, e.g. in peritoneal dialysis- (PD) and infusion fluids.•Clinical effects are caused by their cytotoxicity and activity to modify proteins.•In PD, (p)GDPs may impair residual kidney function and damage the peritoneum.•Studies on the chemistry of (p)GDP formation help to develop mitigation strategies.Carbohydrate degradation products are formed during heat sterilization in drugs containing (poly-)glucose as osmotic agents. Given this situation, peritoneal dialysis fluids (PDFs) and infusion fluids are of particular clinical relevance, because these drugs deliver process contaminants either over a longer period or directly into the circulation of patients who are critically ill. For the development of suitable mitigation strategies, it is important to understand the reaction mechanisms of carbohydrate degradation during sterilization and how the resulting products interact with physiological targets at the molecular level. Furthermore, reliable, comprehensive, and highly sensitive quantification methods are required for product control and toxicological evaluation.