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Background: The complexity of food structure is such as to hinder its inclusion in mathematical models predicting food properties and transformations, although a considerable impulse is being determined by using artificial intelligence. As a matter of fact, food definition currently neglects the structural description, even in those fields for which structure is demonstrated to have a decisive role, such as nutrition. Scope and approach: This review aims to analyse the current knowledge about the structure of foods and its potential use to numerically define the sensory and nutritional quality, as well as the stability properties. Starting from this information, a possible methodology is explored to build, even in an automated way, mathematical models for simulating and predicting the properties of food. A model pipeline has been proposed and applied to pasta, in particular exploiting the description of the structural changes occurring upon cooking. Key findings and conclusions: Foods may be designed in silico, based on automated pipelines for direct extraction of information on rheological and sensory properties as derived from structure images and from data on the dynamic state of the water. The ultimate goal of these approaches is to make more limited use of expensive and time-consuming experiments on physically prepared foods to get to use digital twins of foods designed in the laboratory.

Osteosarcoma is a bone cancer characterized by the production of osteoid tissue and immature bone from mesenchymal cells. Osteosarcoma mainly affects long bones (femur is most frequently site) and occur in children and young adults with greater incidence. Here, we investigated the role accomplished by polydatin, a natural antioxidative compound, in promoting osteogenic differentiation alone or after radiation therapy on osteosarcoma cells. In vitro, polydatin significantly induced cell cycle arrest in S-phase and enhanced bone alkaline phosphatase activity. Moreover, the differentiation process was paralleled by the activation of Wnt-β-catenin pathway. In combination with radiotherapy, the pretreatment with polydatin promoted a radiosensitizing effect on osteosarcoma cancer cells as demonstrated by the upregulation of osteogenic markers and reduced clonogenic survival of tumor cells. Additionally, we analyzed, by mass spectrometry, the secretion of sphingolipid, ceramides, and their metabolites in osteosarcoma cells treated with polydatin. Overall, our results demonstrate that polydatin, through the secretion of sphingolipids and ceramide, induced osteogenic differentiation, alone and in the presence of ionizing therapy. Future investigations are needed to validate the use of polydatin in clinical practice as a potentiating agent of radiotherapy-induced anticancer effects.

Heat-stable peptidases released in refrigerated raw milk by psychrotrophic bacteria are responsible for UHT milk gelation. K-casein-derived caseinomacropeptides, identified by mass spectrometry, were constantly detected in gelled milk by capillary electrophoresis. Strains of Pseudomonas fluorescens, Ps. poae and Chryseobacterium joostei, selected among aprX-positive strains from raw milk, were incubated in milk up to 6 days at 4 degrees C before sterilization (98 degrees C/4 min). Samples were then stored at 25 or 40 degrees C, visually observed for gelation, and analysed for presence of caseinomacropeptides throughout 90 days of storage. Depending on cold pre-incubation time, caseinomacropeptides accumulated well before gelation onset in milk stored at 25 degrees C. Caseinomacropeptides were successively degraded, especially in milk stored at 40 degrees C, due to extensive proteolysis, and an abundant sediment developed instead of a gel. The caseinomacropeptides are here presented as an early indicator of UHT milk gelation and a mechanism explaining this phenomenon is proposed.

In this work, we explored the “deep” seed peanut proteome by using both two dimensional electrophoresis (2- DE)-based analysis run under reducing and non-reducing condition (protein-centric) and LC-MS/MS gel-free proteomic (peptide-centric). The former approach allowed to identify high molecular weight disulfide-linked Ara h 1 and Ara h 3 heteroligomers and Ara h 1 homoligomers linked through covalent bonds other than disulfides. The occurrence of these protein complexes revealed natural interactions between Ara(s) subunits with a possible involvement in the allergenic potential of peanut. The second approach, also referred to as shot-gun proteomics, allowed the identification of 149 gene products, including low-abundance proteins escaped the 2-DE detection. Interestingly, we identified 60 proteins never catalogued previously. The complementary exploitation of two proteomic approaches enabled the access to new relevant information about the complexity of the peanut proteome, with special emphasis to the complement of allergens (allergome).

Hemp (Cannabis sativa L.), traditionally cultivated for industrial use and harvested for fibers and seeds, has raised much interest as a sustainable crop in the last years. Recently, hemp seeds and derived oil have started to be used in a variety of food products. Hemp-based food products are considered less allergenic than those from other edible seeds, although this statement has never been experimentally verified. In this study high purity grade hemp flour (HF) and hemp protein isolate (HPI) were obtained through a fast and cheap process starting from defatted hemp cakes, a residue of hempseed oil extraction. HPI resulted enriched at nearly 86% protein, mainly constituted by the storage protein edestin (accounting for 70% total protein). In vitro protein digestibility was determined using a static model of gastrointestinal digestion (GID), which included a final step with purified brush border membrane (BBM) enzyme preparations. HF and HPI showed a high degree of digestibility. The survival of potential bioactive and/or allergenic peptide sequences in digests was investigated by peptidomic analysis. Only a limited number of sequences survived GID. Among them, fragments from 12 seed proteins. These fragments were precursors of sequences with potential bioactive peptides, which might justify the bioactivity of HPI hydrolysates, reported in previous studies. More importantly, all known hemp allergens, including the major thaumatin-like protein and LTP, were entirely eliminated by the HPI production process, neither fragments of the proteins were present after GID. These data support the use of HPI as an ingredient for hypoallergenic foods.

The demand for sustainably produced proteins is increasing with the world population and is prompting a dietary shift toward plant sourced proteins. Vegetable proteins have lower digestibility and biological value compared to animal derived counterparts. We explored sprouting of chickpea seeds as a strategy for improving digestibility. Protein evolution associated with by the sprouting process was assessed by proteomics. The sprouting induced breakdown of seed storage proteins and doubled the release of free alpha-amino nitrogen in sprouted chickpea flour. During sprouting, several enzymes involved in plant development were newly expressed. An ex vivo model of gastroduodenal and jejunal digestion was applied to assess the bioaccessibility of the protein digests. Proteins from chickpea sprouts showed a greater susceptibility to digestion with a 10% increase in alpha amino nitrogen. Peptides with potential immunoreactivity or bioactivity were catalogued in both digested chickpea sprouts and seeds using an in-silico approach. Peptides belonging to the non-specific transfer proteins, which are allergens in pulses, and peptides belonging to an IgE-binding hemagglutinin protein could only be identified in the digested chickpea sprouts. The observation collected paved the way to immune-based evaluations to assess the effect of germination on the allergenic potential.

Kashk is a fermented dairy product typical of the Middle East, traditionally produced with sour milk and/or dairy waste. The kashk water-soluble peptide fraction was characterized at the molecular level by liquid chromatography-mass spectrometry and its antibacterial and skin healing activity was evaluated. Antibacterial assays showed a significant antibacterial activity against clinical isolates of Staphylococcus aureus (S. aureus) from patients with atopic dermatitis, inhibiting bacterial growth by approximately 45% (500μg/mL). Skin repair activity was evaluated on keratinocytes through scratch tests showing accelerated wound closure in vitro in the presence of TNF-α, by approximately 44% (500μg/mL), compared to control cells. Furthermore, based on the MTT assay, the kashk peptide fraction did not show toxicity on keratinocytes. The results suggested that the peptide kashk extract may be useful in skin care for patients with atopic dermatitis.

Myrtus communis L. (myrtle) is native to the Mediterranean region and Western Asia. Its leaves have demonstrated its potential effect towards different bioactivities like anti-diabetic, anti-diarrheic, anti-ulcer, anti-cancer, among others. These activities have been associated with its phenolic content. In this sense, the aim of this work has been to develop a new pressurized-liquid extraction procedure (PLE), by using a response surface methodology (RSM), to evaluate the phenolic composition from myrtle leaves by HPLC-DAD-TOF-MS. Previously, different solvents such as methanol, ethanol, and acetone/water mixtures were tested by using ultrasound-assisted extraction (UAE) in order to select the most suitable one. Subsequently, a Box-Behnken design (BBD) was performed according to the effect of ethanol/water ratio (50, 75, and 100% (v/v)), temperature (50, 125, and 200 °C), and extraction time (5, 18, and 30 min). The optimal conditions achieved with the established method were 71% ethanol/water, 137 °C, and 19 min. The analysis of the obtained extracts by HPLC-DAD-TOF-MS allowed the characterization of 15 new compounds in myrtle leaves. Finally, high amounts of gallic and ellagic acid were found in the optimized PLE extracts (3.31 ± 0.03 and 3.88 ± 0.09 mg/g leaf dry weight (d.w.), respectively), and PLE reported greater recovery of total phenolic compounds than UAE (30 ± 1 and 22.4 ± 0.6 mg/g leaf d.w., respectively).

The present study aimed to obtain novel information regarding the pathways of a polyphenol-rich coffee silverskin extract (CSE) for inhibiting the formation of advanced glycation products (AGE). Chlorogenic acid (CGA) is the main phenol present in coffee beans. The contribution of CGA to the antiglycoxidative properties of CSE was also evaluated by analysis of CGA standard. The CGA (90%) and CSE (49%) significantly inhibited (p < 0.001) the formation of fluorescent AGE in a protein–methylglyoxal (MGO) model system (37 °C for 96 h). The lack of MGO-derivative AGE in the presence of CGA and CSE was verified using liquid chromatography–tandem mass spectrometry techniques, which evidenced the almost complete absence of arginine adducts after 24 h of reaction. The inhibitory effect of polyphenols present in CSE may be associated to its carbonyl trapping capacity as well as to its ability to react with side-chains of protein amino residues blocking the reaction sites. In conclusion, aqueous CSE can be considered a natural source of various inhibitors of in vitro formation of AGE acting by different pathways., The present research was supported by SUSCOFFEE project (AGL57239-R, 2014). B. Fernandez-Gomez thanks the FPI Ph.D. program of MINECO for supporting her research career (BES-046827, 2011). This investigation was in collaboration Spain-Italy thanks a mobility grant (EEBB-I-14-08220, 2014).

The intestine presides over a series of vital functions in the human body, among which the digestion/absorption of nutrients. Despite their major digestion role, the impact of the enzymes of the luminal intestinal surface on food components has been considered in relatively few experiments of simulated gastrointestinal digestion. In contrast, the identification of proteolitically stable peptides which survived digestion in multiphasal models that also included a step with small intestinal brush border membrane (BBM) peptidases has provided physiologically consistent results. Herein, we critically review the use of BBM enzymes to simulate the intestinal digestion of dietary polypeptides. Addressing the controversial issue of the in vitro-in vivo correspondence of the digestion models, the review emphasizes the need to establish consensus protocols to simulate the intestinal step, for instance using the BBM hydrolases at least in a selected number of cases. The factors that have limited the development of relevant models of intestinal degradationare discussed together with hints to possible alternatives, forthcoming approaches and future perspectives to reproduce the physiopathology of the human small intestine. (C) 2015 Elsevier Ltd. All rights reserved.

Exclusively breast-fed infants can exhibit clear signs of IgE or non IgE-mediated cow's milk allergy. However, the definite characterization of dietary cow's milk proteins (CMP) that survive the maternal digestive tract to be absorbed into the bloodstream and secreted into breast milk remains missing. Herein, we aimed at assessing possible CMP-derived peptides in breast milk. Using high performance liquid chromatography (HPLC)-high resolution mass spectrometry (MS), we compared the peptide fraction of breast milk from 12 donors, among which 6 drank a cup of milk daily and 6 were on a strict dairy-free diet. We identified two bovine ?-lactoglobulin (?-Lg, 2 out 6 samples) and one ?s1-casein (1 out 6 samples) fragments in breast milk from mothers receiving a cup of bovine milk daily. These CMP-derived fragments, namely ?-Lg (f42-54), (f42-57) and ?s1-casein (f180-197), were absent in milk from mothers on dairy-free diet. In contrast, neither intact nor hydrolyzed ?-Lg was detected by western blot and competitive ELISA in any breast milk sample. Eight additional bovine milk-derived peptides identified by software-assisted MS were most likely false positive. The results of this study demonstrate that CMP-derived peptides rather than intact CMP may sensitize or elicit allergic responses in the neonate through mother's milk. Immunologically active peptides from the maternal diet could be involved in priming the newborn's immune system, driving a tolerogenic response.

Type-2 diabetes is continuously increasing worldwide. Hence, there is a need to develop functional foods that efficiently alleviate damage due to hyperglycaemia complications while meeting the criteria for a sustainable food processing technology. Inhibition of mammalian alpha-amylase and alpha-glucosidase was studied for white grape skin samples recovered from wineries and found to be higher than that of the drug acarbose. In white grape skins, quercetin and kaempferol derivatives, analysed by UPLC-DAD-MS, and the oligomeric series of catechin/epicatechin units and their gallic acid ester derivatives up to nonamers, analysed by MALDI-TOF-MS were identified. White grape skin was then used for enrichment of a tomato puree (3%) and a flat bread (10%). White grape skin phenolics were found in the extract obtained from the enriched foods, except for the higher mass proanthocyanidin oligomers, mainly due to their binding to the matrix and to a lesser extent to heat degradation. Proanthocyanidin solubility was lower in bread, most probably due to formation of binary proanthocyanin/protein complexes, than in tomato puree where possible formation of ternary proanthocyanidin/protein/pectin complexes can enhance solubility. Enzyme inhibition by the enriched foods was significantly higher than for unfortified foods. Hence, this in vitro approach provided a platform to study potential dietary agents to alleviate hyperglycaemia damage and suggested that grape skin phenolics could be effective even if the higher mass proanthocyanidins are bound to the food matrix.

Background and Aims Varietal thiols, important odorous molecules in wine, have been identified in Vitis vinifera L. grapes as S-cysteinylated and S-glutathionylated odourless precursors. The aim of this paper is to investigate the presence of these precursor compounds during ripening of two Italian white grape cultivars, Grechetto and Malvasia del Lazio, in comparison with the well-characterised, reference cultivar Sauvignon Blanc. Methods and Results Grapes were sampled and sorted according to density in order to obtain homogeneous samples at the TSS concentration of 16, 19 and 22°Brix, as a synthetic index of ripening stage. After sorting and processing the grapes, the concentration of thiol precursors contained in each sample of juice was determined by liquid chromatography-quadrupole MS. For the first time, S-3-(4-sulfanyl-4-methylpentan-2-ol)-glutathione was identified and quantified in the three cultivars as a glutathione-conjugated precursor. Both S-cysteinylated and S-glutathionylated precursors were ubiquitous in the three cultivars, although their concentration varied significantly. Conclusion Thiol precursors increased with TSS during ripening in Sauvignon Blanc and Grechetto, while no variation of these odourless compounds was observed in Malvasia del Lazio, which also had a lower concentration of these precursors. Significance of the Study This work contributes to defining the significance of these precursors to the varietal attributes of the resulting wines, as well as indicating the optimal harvest time of grapes for maximal expression of thiol varietal attributes in wine.

Wheat beer is a traditional light-colored top-fermenting beer brewed with at least 50% malted (e.g., German Weissbier) or unmalted (e.g., Belgian Witbier) wheat (Triticum aestivum) as an adjunct to barley (Hordeum vulgare) malt. For the first time, we explored the proteome of three Weissbier samples, using both 2D electrophoresis (2DE)-based and 2DE-free strategies. Overall, 58 different gene products arising from barley, wheat, and yeast (Saccharomyces spp.) were identified in the protein fraction of a representative Weissbier sample analyzed in detail. Analogous to all-barley-malt beers (BMB), barley and wheat Z-type serpins and nonspecific lipid transfer proteins dominated the proteome of Weissbier. Several alpha-amylase/trypsin inhibitors also survived the harsh brewing conditions. During brewing, hundreds of peptides are released into beer. By liquid chromatography-electrospray tandem mass spectrometry (LC-ESI MS/MS) analysis, we characterized 167 peptides belonging to 44 proteins, including gliadins, hordeins, and high- and low-molecular-weight glutenin subunits. Because of the interference from the overabundant yeast-derived peptides, we identified only a limited number of epitopes potentially triggering celiac disease. However, Weissbier samples contained 374, 372, and 382 ppm gliadin-equivalent peptides, as determined with the competitive G12 ELISA, which is roughly 10-fold higher than a lager BMB (41 ppm), thereby confirming that Weissbier is unsuited for celiacs. Western blot analysis demonstrated that Weissbier also contained large-sized prolamins immunoresponsive to antigliadin IgA antibodies from the pooled sera of celiac patients (n = 4).

The aim of the present study was to identify and characterize the celiacogenic/immunogenic proteins and peptides released during digestion of pasta (Triticum durum semolina). Cooked pasta was digested using a harmonized in vitro static model of oral-gastro-duodenal digestion. The course of pasta protein digestion was monitored by SDS-PAGE, and gluten proteins were specifically analyzed by Western blot using sera of celiac patients. Among the allergens, nonspecific lipid-transfer protein was highly resistant to gastroduodenal hydrolysis, while other digestion-stable allergens such as alpha-amylase/trypsin inhibitors were not detected being totally released in the pasta cooking water. To simulate the final stage of intestinal degradation, the gastroduodenal digesta were incubated with porcine jejunal brush-border membrane hydrolases. Sixty-one peptides surviving the brush-border membrane peptidases were identified by liquid chromatography-mass spectrometry, including several gluten-derived sequences encrypting different motifs responsible for the induction of celiac disease. These results provide new insights into the persistence of wheat-derived peptides during digestion of cooked pasta samples.

The aim of this work was to assess whether the characteristic polyphenol traits of cherry biotypes persisted in thermally processed cherry products, such as jam. Thus, the RP-HPLC-diode array detector profiles of both colorless polyphenols and anthocyanins from three cherry varieties (two sweet and one tart cherry) were compared with those of low-sugar jam sourced from the same cultivars. Individual components were characterized by mass spectrometry. The total phenolic and total anthocyanin content as well as the radical scavenging potential (residual 75–91, 88–91 and 73–75%, respectively) were only slightly reduced by deep thermal treatments. Apart from the interconversion among the isomers of chlorogenic acid, the profile of both colorless polyphenols and anthocyanins substantially survived the jam manufacturing under conventional temperature–time regimen (80 °C, 1 h). The species- and cultivar-specific polyphenol molecular asset, especially the anthocyanin pattern, has potential to be monitored for traceability purpose, aimed to the varietal assessment of cherry biotypes used for producing jam.

Structural changes ensuing from the non-covalent absorption of bovine beta-lactoglobulin (BLG) on the surface of polystyrene nanoparticles were investigated by using spectroscopic approaches, by assessing the reactivity of specific residues, and by limited proteolysis/mass spectrometry. Also, the immunoreactivity of absorbed and free BLG was compared. All these approaches indicated substantial rearrangements of the protein structure in the absorbed state, in spite of the reported structural rigidity of BLG. Changes made evident by experimental measurements were confirmed by computational approaches. These indicate that adsorption-related changes are most marked in the area between the main C-terminal alpha helix and the beta-barrel, and lead to full exposure of the thiol on Cys121, consistent with experimental measurements. In the computational model of bound BLG, both Trp61 and Trp19 also move away from their neighboring quenchers and become solvent-exposed, as indicated by fluorescence measurement. Upon binding, the beta-barrel also loosens, with a substantial increase in immunoreactivity and with noticeable changes in the trypsinolytic pattern. The possible general significance of the structural changes reported here for non-covalently adsorbed BLG is discussed with respect to recognition events involving surface-bound proteins, as are aspects related to the carrier function(s) of BLG, and to its use as a common ingredient in many food systems. Proteins 2014; 82:1272–1282. © 2013 Wiley Periodicals, Inc.

High resolution mass spectrometry was employed to study a bloom of the Cyanobacterium Planktothrix rubescens in a dam for drinking water on Lake Occhito, near the town of Foggia, in Southern Italy, to characterise the cyanotoxins produced and to evaluate the related health risks for consumers. The cyanotoxin production in various sites of the contaminated dam was studied by MALDI-TOF mass spectrometry and liquid chromatography coupled to Q/TOF tandem mass spectrometry analysis of water and cyanobacteria samples. MALDI-TOF mass spectrometry was employed for rapid screening over a wide mass range; liquid chromatography coupled to Q/TOF tandem mass spectrometry allowed for molecular structure confirmation of each single cyanotoxin. This analytical strategy allowed for profiling the cyanotoxins produced during the bloom and calculating the concentrations of the main contaminants in drinking water. Microcystins, anabaenopeptins and aeruginosins were the main cyanotoxins identified. A des-methyl variant of the microcystin-RR was recognised as the most abundant compound in all water samples; liquid chromatography coupled to Q/TOF tandem mass spectrometry allowed for molecular structural confirmation of detected substances, and quantitative analysis of microcystins. The contamination levels over 3 weeks were monitored, by sampling from different sites of the dam. This analytical strategy allows for complete characterisation of the cyanotoxins produced during this kind of environmental outbreaks, for time-dependent monitoring of pollution levels and thus represents a powerful tool to prevent intoxication for consumers of contaminated drinking water collected from the dam.

A "virtual two-dimensional (2D)-like" approach based on pH 2.5-6.0 gel isoelectrofocusing coupled to MALDI-TOF mass spectrometry (MS) was addressed for challenging the ovine casein heterogeneity due to polymorphism and post-translational modifications. The procedure enabled the identification of the main casein components including i) the four casein families at different degrees of phosphorylation, ii) nonallelic variants of either alpha(s1)- or alpha(s2)-CN, iii) differently glycosylated kappa-CN, and iv) high molecular mass proteolytic peptides. Protein assignment was confirmed by MALDI-TOF (MS) mass mapping of the tryptic digests, while phosphorylation sites were localized by tandem mass spectrometry. A straightforward identification was hindered by unexpected molecular mass shifts due to the oxidization of alpha(s1)- and the beta-CN. Indeed, using nano-ESI-MS, the casein sub-types were proved to contain a variable number of oxidized methionine residues. Data were additionally substantiated using polyclonal antibodies raised against the single casein families for immunolabelling purposes. The outcomes herein demonstrate the potentiality of the "virtual 2D-like" approach and provide indications useful for the inter-laboratory discrepancies in casein identification. They also greatly facilitate the comparison of data and the establishment of multi-user image-based isoelectrofocusing gels. (C) 2012 Elsevier Ltd. All rights reserved.

Among the methods that have been developed for anthocyanin characterization, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) offers several analytical advantages in terms of speed, minimal sample handling, specificity, and reliability, without requiring any previous chromatographic separation. This study used MALDI-TOF MS to profile the anthocyanins from the berry skins of 23 red grape varieties clustered as (i) authentic Vitis vinifera grapes, (ii) American hybrid cultivars, and (iii) Casavecchia cultivars, previously characterized as functional crosses of V. vinifera with nondefined hybrid grapevines. Anthocyanin profiling demonstrated evidence of several varietal traits that enabled the differentiation of authentic V. vinifera from hybrid cultivars on a molecular basis. In particular, acyl 3,5-O-diglucoside anthocyanins were established as easily monitored molecular markers of the hybrid varieties. It was also demonstrated that MALDI-post source decay MS is a powerful tool to differentiate isobaric 3,5-O-diglucosides and their derivatives, which prevail in hybrid cultivars, from acylated 3-O-glucoside anthocyanins.

The composition of milk has adapted during the evolution of the species to fulfill the specific nutritional needs of the offspring. Currently, it is widely recognized that milk benefits go beyond mere nutrition and serve as a source of a number of functional components to the newborn, particularly host defense effectors. However, the human milk proteome description is still incomplete, primarily because the detection of low-abundance proteins remains challenging. To overcome the limitations of the classical electrophoresis-based approach, previously separated milk fat globule membrane (MFGM) and whey protein fractions were analyzed by nanoflow-high performance liquid chromatography (HPLC)/Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometry (MS). This shotgun strategy showed an as yet unmatched potential to profile low-abundance proteins in human milk. Proteins associated with 301 different gene products were identified, some of which could be clustered into subsets of protein isoforms, thus providing one of the largest protein inventories of human milk. The identified proteins, which were derived from multiple metabolic pathways, are involved in different physiological functions, such as membrane trafficking, cell signaling, fat metabolism and transport, metabolite delivery, protein synthesis/proteolysis or folding, and immunity-related actions. Nevertheless, it appears clear from this study that the overall picture of the human milk proteome is still incomplete, although several protein signatures of milk evolution are emerging.

IgE-mediated allergy to soybean may be connected to an incomplete protein digestion causing an inappropriate immune response in the gut. The aim of this study was to gain insight on the digestibility and immunoreactivity of β-conglycinin and its deglycosylated form. To achieve this goal both proteins were digested mimicking gastrointestinal physiological conditions. Digests were characterised by SDS-PAGE, MALDI-TOF-MS and immunological assays. Data on IgG binding western blot provided key information related to the composition of the digests indicating that whereas deglycosylated β-conglycinin was completely removed by gastrointestinal digestion, α and β subunits of β-conglycinin glycoprotein partially survive the process of digestion. Such differential digestive behaviour might be influenced by glycan moieties, at least to some extent, and should be an additional factor to be considered on its potential allergenicity. Results also support the existence of linear epitopes in the three subunits (α, α′, and β) of both protein forms. © 2011 Elsevier Ltd. All rights reserved., This work has been funded by ERDF-co-financed grant AGL2007-60007/ALI, CONSOLIDER Ingenio 2010 FUN-C-FOOD: CSD 2007-00063, ALIBIRD-CM-S0505/AGR-0153 and IT-2009- 0087 projects. Dr. M.A-B. thanks European Union COST-Action 927 for Short Term Scientific Mission and Danone Institute for her fellowship.

Bovine β-casein and its C-terminal sequence (191–209) were previously shown to possess immunomodulatory properties in studies on partially purified peptides from hydrolysates, where minor amounts of contaminants may affect the cellular response. The inhibitory effect of β-casein and of eight C-terminus synthetic β-casein peptides on mitogen-induced spleen cell proliferation was compared. Use of synthetic peptides allowed the unambiguous and accurate identification that a seven amino acid sequence at the C-terminus, including a PFP motif, was sufficient for immunomodulation. Substitution of the last proline (P206) in the PFP motif with D-Pro had a negative impact on the immunosuppressory activity of all these short peptides, whereas substitution of P206 with structural analogues of proline had almost no impact. A relationship was found between the immunomodulatory properties and the structural features of these peptides, as assessed by various spectroscopic approaches, indicating a role of structure in eliciting the immunomodulatory activity of these peptides.

In recent years, the occurrence of cyanobacterial blooms in eutrophic freshwaters has been described all over the world, including most European countries. Blooms of cyanobacteria may produce mixtures of toxic secondary metabolites, called cyanotoxins. Among these, the most studied are microcystins, a group of cyclic heptapeptides, because of their potent hepatotoxicity and activity as tumour promoters. Other peptide cyanotoxins have been described whose structure and toxicity have not been thoroughly studied. Herein we present a peptidomic approach aimed to characterise and quantify the peptide cyanotoxins produced in two Italian lakes, Averno and Albano. The procedure was based on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry mass spectrometry (MALDI-TOF-MS) analysis for rapid detection and profiling of the peptide mixture complexity, combined with liquid chromatography/electrospray ionisation quadrupole time-of- flight tandem mass spectrometry (LC/ESI-Q-TOF-MS/MS) which provided unambiguous structural identification of the main compounds, as well as accurate quantitative analysis of microcystins. In the case of Lake Averno, a novel variant of microcystin-RR and two novel anabaenopeptin variants (Anabaenopeptins B(1) and Anabaenopeptin F(1)), presenting homoarginine in place of the commonly found arginine, were detected and characterised. In Lake Albano, the peculiar peptide patterns in different years were compared, as an example of the potentiality of the peptidomic approach for fast screening analysis, prior to fine structural analysis and determination of cyanotoxins, which included six novel aeruginosin variants. This approach allows for wide range monitoring of cyanobacteria blooms, and to collect data for evaluating possible health risks to consumers, through the panel of the compounds produced along different years.

In this study, the protein and peptide fractions of two commercial Italian barley malt beers, made with different processes by the same producer, have been analysed with a combined immunochemical and mass spectrometry approach. The “gluten” content of beer samples, measured with the R5 monoclonal antibody, was below the caution limit proposed by the Codex Alimentarius for gluten-free foods. The proteomic approach allowed to identify a 17 kDa avenin-like protein partially homologous to hordeins, that was particularly abundant in foam, in addition to the already reported barley albumins (Z 4 -barley and ns-LTPs) and to minor amounts of yeast glycolytic enzymes. No intact hordeins were detected, although fragments derived from γ 3 - and B-hordein were present. In consideration of the many implications of the protein/peptide pattern, these data provide useful information to improve quality and safety of beer.

Owing to its extensive use in the human diet, wheat is among the most common causes of food-related allergies and intolerances. Allergies to wheat are provoked by ingestion, inhalation or contact with either the soluble or the insoluble gluten proteins in wheat. Gluten proteins, and particularly the gliadin fraction, are also the main factor triggering celiac disease, a common enteropathy induced by ingestion of wheat gluten proteins and related prolamins from oat, rye and barley in genetically susceptible individuals. The role of gliadin and of its derived peptides in eliciting the adverse reactions in celiac disease are still far from being completely explained. Owing to its unique pathogenesis, celiac disease is widely investigated as a model immunogenetic disorder. The structural characterization of the injuring agents, the gluten proteins, assumes a particular significance in order to deepen the understanding of the events that trigger this and similar diseases at the molecular level. Recent developments in proteomics have provided an important contribution to the understanding of several basic aspects of wheat protein-related diseases. These include: the identification of gluten fractions and derived peptides involved in wheat allergy and intolerance, including celiac disease, and the elucidation of their mechanism of toxicity; the development and validation of sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for intolerant patients; and the formulation of completely new substitute foods and ingredients to replace the gluten-based ones. In this article, the main aspects of current and prospective applications of mass spectrometry and proteomic technologies to the structural characterization of gluten proteins and derived peptides are critically presented, with a focus on issues related to their detection, identification and quantification, which are relevant to the biochemical, immunological and toxicological aspects of wheat intolerance.

Ovine α s1 -casein ( CSN1S1 ) allele I ( CSN1S1 ∗ I ) was characterized at the molecular genetic and protein level. Sequencing of CSN1S1 cDNA and mature protein showed the absence of exon 7 from CSN1S1 ∗ I in comparison with the C ″ genetic variant of the C phenotype. This allelic aberration is correlated with a sequence difference in 5′-splice donor sequence of intron 7 (g.656T > A), leading to upstream skipping of exon 7. Consequently mRNA sequence of ovine CSN1S1 ∗ I is 24 bp shorter than complete coding sequence leading to an abbreviation of eight amino acids in the mature protein, resulting in a lower degree of phosphorylation in comparison with CSN1S1 ∗ C ″. However CSN1S1 ∗ I was expressed at a quantitative level similar to that for the C ″ reference variant. Using amplified created restriction site polymerase chain reaction, a DNA-based test for identification of CSN1S1 ∗ I was developed. Altogether six nucleotide substitutions were identified within intron 6 and intron 7 of CSN1S1 variants, forming three different haplotypes.

Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

β-Conglycinin is a functional glycoprotein and one of the most important soybean allergens. The aim of the present research was to investigate the role of the N-glycans moieties of β-conglycinin on its in vitro immunoreactivity. The soy allergen was obtained by isoelectric precipitation from commercial soy protein isolate and was enzymatically deglycosylated by PNGase F (Peptide N-Glycosidase F EC 3.5.1.52). In order to optimize deglycosylation conditions different reaction times and allergen concentrations were tested. The extent of deglycosylation was estimated by SDS–PAGE, CZE, RP-HPLC, and MALDI-TOF MS analyses, which provided information related to changes in protein structure. The antigenicity of both native β-conglycinin and its deglycosylated form was evaluated by western-blotting and indirect ELISA employing polyclonal rabbit anti-soybean sera and horseradish peroxidase-labeled goat anti-rabbit IgG while the in vitro allergenicity was assessed by means of indirect competitive inhibition ELISA employing human sera (IgE) of soy allergics. β-Conglycinin was effectively deglycosylated by PNGase F. Data on immunological tests suggested that glycosyl moieties forming this glycoprotein might be involved in its immunoreactivity.

The formation of adducts between acrylamide and hemoglobin in vitro was investigated by using mass spectrometric methodologies to identify the amino acid residues sensitive to alkylation. Liquid chromatography-electrospray ionisation mass spectrometry analysis of either intact or trypsin-digested alpha- and beta-globin chains isolated from hemolysate samples incubated in vitro with acrylamide at different molecular ratios allowed us to identify Cys93 of beta-globin as the most reactive site in hemoglobin, according to a Michael-type addition reaction between acrylamide and the sulphydryl group of cysteine. The only other reactive sites were Cys104 of alpha-globin and the N-terminal amino groups of both chains. The method developed, based on electrospray ionisation quadrupole time-of-flight tandem mass spectrometry analysis of intact globin chains was able to specifically detect low levels of adducts. In this way, rapid identification of alkylated portion of Hb was achieved to be potentially used as a biomarker for high-sensitivity biological monitoring.

Molecular approaches by means of a combined use of mass spectrometric techniques can be important in order to open new possibilities in the differentiation and defense of typical products; in this study, a possible approach to the analysis of varietal volatile compounds and some precursors of a non-aromatic grape variety (Falanghina cv., Vitis vinifera L.) was traced through a combined use of techniques based on mass spectrometry (GC/MS, LC/ESI-MS, MALDI-TOF-MS). Dominant terpene compounds (limonene, cis-furanlinalool oxide, geraniol, 4-carene, myrcene, linalool, a-terpineol), terpene-derivatives (bornyl acetate, menthol), terpene glycosides (glucosides, arabinosylglucosides and rhamnosylglucosides of linalool and geraniol), and norisoprenoids (b-damascenone) were identified in grapes and monovarietal wines, overcoming the analytical difficulties deriving from the low concentration of these compounds strictly related to the variety. The potential release of varietal volatile compounds from the grapes was also explored by enzyme hydrolysis.

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 μm native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme–electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.

The main peptides produced by hydrolysis of water buffalo beta-casein with plasmin were characterized by capillary electrophoresis and mass spectrometry and compared with their bovine homologous. A novel breakdown product arising from the hydrolysis of water buffalo beta-casein, originated by the presence of a plasmin-sensitive Lys bond at position 68 was identified, which was not present in bovine beta-casein. On the basis of this evidence, an improved procedure for the detection and the differentiation of the products of plasmin hydrolysis of bovine and water buffalo beta-casein by capillary isoelectric focusing was set-up. In the experimental conditions, the gamma-casein from the two species was efficiently separated. Comparison of the capillary electropherograms with those obtained by ultra-thin-layer isoelectric focusing, the reference method for routine analysis of plasmin digests of casein, suggests that capillary electrophoresis isoelectric focusing may constitute a successful alternative to the traditional slab gel electrophoresis analysis of plasmin digests of casein either for basic structural studies or for applications in the quality assessment of dairy products.

The relationship between iron uptake by aporubredoxins (apoRds) and formation of native holorubredoxins (holoRd), including their Fe(SCys)(4) sites, was studied. In the absence of denaturants, apoRds exhibited spectroscopic features consistent with structures very similar to those of the folded holoRds. However, additions of either ferric or ferrous salts to the apoRds in the absence of denaturants gave less than 40% recovery of the native holoRd circular dichroism and UV-vis spectroscopic features. In the presence of either 6 M urea or 6 M guanidine hydrochloride, the nativelike structural features of the apoRds were absent. Nevertheless, nearly quantitative recoveries of the native holoRd spectroscopic features were achieved by addition of either ferric or ferrous salts to the denatured apoRds without diluting the denaturant. Consistent with this observation, the native spectroscopic features were unaffected by addition of the same denaturant concentrations to the as-isolated holoRds. Denaturing concentrations of urea or guanidine hydrochloride also increased the rates of holoRd recoveries from apoRds and ferrous salts. Mass spectrometry confirmed that ferric iron binding to the denatured apoRds precedes the recoveries of protein secondary structures and Fe(SCys)(4) sites. Thus, iron binding to the apoRds guides, both kinetically and thermodynamically, refolding to the native holoRd structures. Our results imply that the ferrous oxidation state would more efficiently drive formation of the native holoRd structure from the nascent apoprotein in vivo, but that the Fe(SCys)(4) site must attain the ferric state in order to achieve its native structure.

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. The human isoform 2 of FADS (hFADS2), which is the product of FLAD1 gene, was over-expressed in Escherichia coli as a T7-tagged protein and identified by MALDI-TOF MS analysis. Its molecular mass, calculated by SDS-PAGE, was approx. 55 kDa. The expressed protein accounted for more than 40% of the total protein extracted from the cell culture; 10% of it was recovered in a soluble and nearly pure form by Triton X-100 treatment of the insoluble cell fraction. hFADS2 possesses FADS activity and has a strict requirement for MgCl2, as demonstrated in a spectrophotometric assay. The purified recombinant isoform 2 showed a kcat of 3.6 x 10(-3)s(-1) and exhibited a KM value for FMN of about 0.4 microM. The expression of the hFADS2 isoform opens new perspectives in the structural studies of this enzyme and in the design of antibiotics based on the functional differences between the bacterial and the human enzymes.

The EU subsidises the use of skimmed-milk powder (SMP) in compound feeding stuffs. There are indications of falsified SMP content due to the addition of plant proteins. These proteins are not allowed in SMP and cannot be identified by the official reference method. Since soy and pea proteins are most likely to be added to SMP, manufactured SMP containing 1 and 5% of these plant proteins was used to develop a sensitive protein identification method based on mass spectrometry (MS). The method included a pre-fractionation step to enrich for plant proteins by using a borate buffer. A very fast perfusion liquid chromatography method including sensitive and selective intrinsic fluorescence detection was developed for monitoring and quantifying the efficiency of the pre-fractionation and screening for plant proteins. After tryptic digestion of the enriched fraction from manufactured adulterated SMP, numerous peptides originating from the major seed proteins of soy (glycinin, beta-conglycin) and pea (legumin, vicilin) could be identified by MS/MS analysis on a quadrupole time-of-flight MS instrument.

We localized the site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg). hTg was chromatographically separated into chondroitin 6-sulfate-containing (hTg-CS) and chondroitin 6-sulfate-devoid (hTg-CS0) molecules on the basis of their d-glucuronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6%. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondroitin 6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxyl-terminal region, starting at residue 2514. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as LTAGXGLRE (residues 2726-2734, X being Ser2730 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5 ′-triiodothyronine (T3) (171%) and 3,5,3′,5′-tetraiodothyronine (T4) (134%) than hTg-CS0. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, in particular, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Lys2714-Gly2715 from proteolysis, during the limited digestion of hTg-CS with trypsin. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg. The potential implications in the ability of hTg to function as an autoantigen and into the pathogenesis of thyroidal and extra-thyroidal manifestations of autoimmune thyroid disease are discussed.

A peptidomics approach was developed to identify transglutaminase-susceptible Q residues within a pepsin-trypsin gliadin digest. Based on tagging with a monodansylcadaverine fluorescent probe, six alpha/beta-, gamma-gliadin, and low molecular weight glutenin peptides were identified by nanospray tandem mass spectrometry. In functioning as an acyl acceptor, tissue transglutaminase was able to form complexes with the glutamine-rich gliadin peptides, whereas by lowering pH, the peptides were deamidated by transglutaminase at the same Q residues, which were previously transamidated. The main common feature shared by the peptides was the consensus sequence Q-X-P. Our findings offer relevant information for the understanding of how dietary peptides interact with the host organism in celiac disease.

Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including αs1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro αs1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.