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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 contribution of cysteine and glutathione conjugates to the formation of thiol compounds in Grechetto wine has been evaluated for the first time, by the use of quantitative GC–MS analysis and odour activity values, as a function of the training system (either Royat cordon or simple Guyot) that was used for the vines. The concentration of conjugate precursors in the grape juice, in the case of both training systems, was found to increase significantly until 22°Brix, except for glutathionylated precursor of 4-methyl-4-sulfanyl-pentan-2-one. The amount of both thiol precursor forms was systematically higher for the Royat cordon system that seems to improve the accumulation of thiol precursors. However, this high juice concentration did not reveal an high content of volatile thiols in derived wine. 4-Methyl-4-sulfanyl-pentan-2-one (4MSP), 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHA) have been identified as aroma compounds in both two monovarietal white wines from Grechetto grape, but only the amounts of 4MSP and 3SHA turned out to be considerably higher than their respective perception thresholds. Considering the growing interest in the varietal wines characteristics on the part of consumers, the results of present work could be useful to improve the scientific knowledge about the relationships between the grape content and wine composition.

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.

Enterotoxigenic Escherichia coli (ETEC) is the main bacterial cause of dehydrating infant diarrhoea in less-developed countries. Labile toxin (LT) is the major virulent factor of ETEC. Easy diagnostic tests are necessary to reduce the number of cases. Immunological methods have some drawbacks and also have important limitations. For that reason, a Liquid Chromatography coupled to UV detector technique (LC-UV) has been optimize to a rapid identification and quantification of LT from bacteria cultures. It is also important to know optimal conditions for LT and with this purpose several enterotoxigenic E. coli strains have been studied to determine the influence of glucose concentration and different culture media on LT production. LC-UV technique demonstrated to be a good method for LT quantification showing values of 15 ng/mL and 45 ng/mL for limits of detection and quantification respectively. LT quantification revealed that toxin production is directly related to the concentration of glucose added in the broth medium. Tryptic Soy Broth is the most efficient culture medium for E. coli growth and enterotoxin production.

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.

Plant-derived insoluble proteins (wheat gluten, and isolates from pea, lentil, and soybean) were used as fining agents in model white wine (made from Catalanesca grapes) after cold stabilization. Plant proteins were effective in giving a fast and remarkable decrease in turbidity. GC/MS and HPLC/MS approaches indicated that individual proteins had a different impact on the levels of compounds relevant to wine stability. Protein stability of wine was not affected by fining with plant proteins. Lentil proteins and gluten gave the best removal of monomeric and dimeric flavonol. Both caused a decrease in the total content of fermentative aroma compounds, such as ethyl esters, acetate esters, and alcohols. Lentil proteins had the highest impact on the aroma components, giving a marked decrease in aroma components. Gluten may thus be regarded as giving the best balance between fining efficacy and retention of aroma compounds. Also, gluten in the treated wines remained well below the suggested threshold for gluten-free foods. This study provides a methodological frame for thorough characterization of the impact of specific interventions on key wine components.

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.

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.

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 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.

Despite several studies support the functionality of casein peptides in vitro, a gap of knowledge still exist about their bioavailability. BIOACTIVE PEPTIDES; MASS-SPECTROMETRY; HEALTH-BENEFITS; BETA-CASEIN; PHOSPHOPEPTIDE; DIGESTION; MILKIn this pilot study the bioavailability of phosphopeptides (CPPs) was investigated in four healthy subjects who consumed for one week 100 g/day of Parmigiano Reggiano cheese after one week of a dairy products-free diet. CPPs were detected in plasma samples from fasting subjects after the cheese-enriched diet and peptides were detected variously in almost all the 4 samples. Some alpha(s1)- and alpha(s2)-CN-derived CPPs, such as alpha(s1)-CN (f43-52 and f43-50) and alpha(s2)-CN (f8-12), (f7-12) and (f6-12) as well as four non-phosphorylated peptides belonging to the C-terminal end of beta-CN (f193-209, f194-209, f200-209) were detected in plasma samples submitted to extraction and enrichment by hydroxyapatite (HA) chromatography followed by MALDI-TOF and nano LC-ESI/MS/MS analysis. Data indicated that casein oligopeptides are bioavailable after a continued intake of cheese. Future studies are warranted to ascertain this finding on a wider population and to clarify the mechanisms behind CPPs bioaccessibility and absorption.

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.

The methodologies of food analysis have greatly evolved over the past 100 years, from basic assays based on solution chemistry to those relying on the modern instrumental platforms. Today, the development and optimization of integrated analytical approaches based on different techniques to study at molecular level the chemical composition of a food may allow to define a 'food fingerprint', valuable to assess nutritional value, safety and quality, authenticity and security of foods. This comprehensive strategy, defined foodomics, includes emerging work areas such as food chemistry, phytochemistry, advanced analytical techniques, biosensors and bioinformatics. Integrated approaches can help to elucidate some critical issues in food analysis, but also to face the new challenges of a globalized world: security, sustainability and food productions in response to environmental world-wide changes. They include the development of powerful analytical methods to ensure the origin and quality of food, as well as the discovery of biomarkers to identify potential food safety problems. In the area of nutrition, the future challenge is to identify, through specific biomarkers, individual peculiarities that allow early diagnosis and then a personalized prognosis and diet for patients with food-related disorders. Far from the aim of an exhaustive review of the abundant literature dedicated to the applications of omic sciences in food analysis, we will explore how classical approaches, such as those used in chemistry and biochemistry, have evolved to intersect with the new omics technologies to produce a progress in our understanding of the complexity of foods. Perhaps most importantly, a key objective of the review will be to explore the development of simple and robust methods for a fully applied use of omics data in food science.

The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization–mass spectrometry (ESI–MS) and matrix-assisted laser desorption ionization–time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HPLC into four major peaks. With ESI–MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography–ESI–MS allowed us to determine each fraction's composition by detecting thirteen αs1-, eleven αs2-, seven β-, and three κ-casein (CN) components. The αs1-CN and αs2-CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences; they were also discretely phosphorylated as κ-CN and β-CN. By CE at pH 2·5, each casein fraction was as heterogeneous as that resulting from ESI–MS for the single HPLC-derived fractions. The separation of αs1-CN and αs2-CN proved to be excellent, with the exception of a co-migration of κ0-CN with a minor αs1-CN component and of a glycosylated κ-CN form with low-phosphorylated αs1-CN and β-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI–MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative αs1-CN variants, the non-allelic αs1-CN and αs2-CN forms, dimeric κ-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.

The protein composition of natural rennet and of chromatographic and crystalline chymosin preparations has been defined by on-line reverse-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (RP-HPLC/ESI-MS) and by tandem mass spectrometry (MS/MS). Natural rennet was found to consist of six chymosin species, corresponding to chymosin A and B genetic variants, each of which comprised a mixture of two other forms differing at theN-terminal end, with one being three residues longer, and the other two residues shorter, than the mature chymosin. Two main tissue proteins were also identified as lysozyme (isozyme 2 plus a novel isozyme labelled 4) and bovine serum albumin. In addition to the proteins, chymosin fragments 247–323 and 288–323 were consistently present in natural rennet. Conversely, chromatographic and crystalline chymosin preparations lacked bovine serum albumin and/or lysozyme, although they contained the same six chymosin species as natural rennet. Since these tissue-specific contaminating proteins each possess specific functions in terms of stabilising enzyme solutions and protecting proteins from proteolytic enzymes, oxidising agents and bacterial proliferation, the rennet may be considered as a functional enzyme preparation that is effectively and naturally adapted to the purposes of cheesemaking. In practice, the highly complex protein composition inherent to natural rennet provided the possibility to differentiate the natural product from other bovine chymosin-based milk-clotting preparations examined in this work. Copyright © 2001 John Wiley & Sons, Ltd.

The present study reports a procedure developed for the identification and quantitative analysis of the adducts formed by interaction of methyl bromide with human hemoglobin, based on combined analysis by electrospray mass spectrometry and automated Edman degradation of either intact globin chains or tryptic peptides of globin chains. The procedure has allowed identification of the reactive sites in human hemoglobin, and has been applied to the analysis of samples modified in vitro by methyl bromide. The results obtained represent the basis for the complete structural characterization of the modified hemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of the degree of alkylation and the identification of modified amino acids in proteins.

The microheterogeneity of trichorzianine-A (TA) from Trichoderma harzianum was studied using single-stage mass spectrometry and high-energy collision-induced dissociation (CID) tandem mass spectrometry. Based on the electrospray ionization and the liquid secondary ion single-state mass spectra, the main components with eight different molecular masses were identified in the mixture. Five contain phenylalaninol and three a tryptophanol C-terminal amino alcohol residue. The tandem mass spectra taken on the protonated molecular ion of these components (MH+) show that most of them contain one main and one or more minor sequence analogues. For the microheterogeneity characterization of this mixture, a previously described method was used based on high-energy CID studies of the different MH+, y7, b12 ions and their satellite ions with ±14 and ±28 mass number differences. In addition to the nine known sequence variants, five new trichorzianines were sequenced from the mixture. It was found, furthermore, that amino acid exchange can occur not only at positions 5, 14, 16 and 19 but also at positions 2, 3, 4, 7, 8 and 9. © 1998 John Wiley & Sons, Ltd.

The comprehensive analysis of natural lipid mixtures is often challenging due to the simultaneous occurrence of functionally and structurally heterogeneous compounds. Modern analytical approaches in system-wide lipidomics essentially rely on mass spectrometry (MS). The overabundant amount of triacylglycerols (TAGs) in most samples hinders the direct detection of phospholipids (PLs) or other low-abundance lipids; therefore, a fractionation step is most often required to reduce sample complexity prior to MS analysis. In this work, we explore the use of hydroxyapatite (HAP) as a chromatographic stationary phase (gel HAP) or chemo-affinity sorbent material (ceramic HAP) to selectively enrich PLs and polar lipids from chicken egg yolk and buttermilk. Due to the affinity of phosphate-containing compounds for HAP, both in-column and in-batch HAP-based chromatography were effective to deplete TAGs before releasing PLs with an eluting ternary system containing sodium phosphate as the displacing agent. Buttermilk gangliosides and PLs co-eluted, indicating that HAP also exhibits a high affinity for sialylated glycolipids and that polar lipids are retained through a combined mechanism of chemo-affinity and hydrogen bonding. To the best of our knowledge, this is the first report in which HAP is proposed as an alternative stationary phase for separating both the PLs and sialylated glycolipids from TAGs in complex lipidomes. The HAP-based chromatography has potential to be improved for the separation of the polar lipid classes. (C) 2014 Elsevier B.V. All rights reserved.

A fragment of bovine thyroglobulin encompassing residues 1218-1591 was prepared by limited proteolysis with thermolysin and continuous-elution polyacrylamide gel electrophoresis in SDS. The reduced and carboxymethylated peptide was digested with endoproteinase Asp-N and fractionated by reverse-phase high performance liquid chromatography. The fractions were analyzed by electrospray and fast atom bombardment mass spectrometry in combination with Edman degradation. The post-translational modifications of all seven tyrosyl residues of the fragment were characterized at an unprecedented level of definition. The analysis revealed the formation of: 1) monoiodotyrosine from tyrosine 1234; 2) monoiodotyrosine, diiodotyrosine, triiodothyronine (T3), and tetraiodothyronine (thyroxine, T4) from tyrosine 1291; and 3) monoiodotyrosine, diiodotyrosine, and dehydroalanine from tyrosine 1375. Iodothyronine formation from tyrosine 1291 accounted for 10% of total T4 of thyroglobulin (0.30 mol of T4/mol of 660-kDa thyroglobulin), and 8% of total T3 (0.08 mol of T3/mol of thyroglobulin). This is the first documentation of the hormonogenic nature of tyrosine 1291 of bovine thyroglobulin, as thyroxine formation at a corresponding site was so far reported only in rabbit, guinea pig, and turtle thyroglobulin. This is also the first direct identification of tyrosine 1375 of bovine thyroglobulin as a donor residue. It is suggested that tyrosyl residues 1291 and 1375 may support together the function of an independent hormonogenic domain in the mid-portion of the polypeptide chain of thyroglobulin.

Les peptides solubles dans le TCA 12 % de lactoserum doux provenant de la fabrication de fromages Provolone et Grana Padano, ainsi que de lactoserum acide provenant de la fabrication de fromage Quarg ont ete separes par CLHP et identifies en combinant la spectrometrie de masse et la degradation d'Edman. Tous les lactoserums contenaient les deux formes phosphorylees du caseinomacropeptide (CMP). Les lactoserums doux contenaient les peptides β-CN(1-29), β-CN(1-28), α s1 -CN(f1-22) et α s1 -CN(f1-23). Ces derniers etaient absents du lactoserum acide, qui presentait neanmoins de nombreux peptides de taille inferieure, signe d'une activite proteasique des bacteries lactiques du levain. La coupure des liaisons peptidiques Phe 23 -Phe 24 et Arg 22 -Phe 23 a egalement ete observee au cours de l'hydrolyse in vitro de la caseine α s1 par la chymosine. Les peptides isoles du lactoserum acide comprenaient 50 peptides issus de la caseine β, 34 de la caseine κ, 7 de la caseine α s1 et aucun de la caseine α s2 . La caseine α s1 etait moins hydrolysee que les caseines β ou κ. Plusieurs peptides formes au cours de l'action de proteinases de type P I - et P III - sur les fractions purifiees de caseines ont ete retrouves aussi dans le lactoserum acide de Quarg. Le mecanisme global de formation de ces peptides dans le lactoserum serait une formation de produits primaires resultant de la degradation des caseines par la chymosine et la plasmine, suivie de la degradation de ces produits par les proteases associees aux enveloppes cellulaires des bacteries lactiques mesophiles du levain. Les resultats montrent aussi que la composition peptidique du lactoserum de Quarg est plus equilibree que celle de deux autres lactoserums et pourrait etre plus favorable a une croissance rapide des bacteries lors de l'utilisation du lactoserum pour un repiquage de levain lactique.

Transport across monolayers of human colon adenocarcinoma (Caco-2) cells represents one of the most widely accepted in vitro models of the intestinal uptake of nutrients and drugs. Herein, the entire panel of peptides produced from caseins (CN) and whey proteins (WP) that survive in vitro sequential gastro-pancreatic digestion and translocate across the Caco-2 monolayers have been characterized by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Except for traces of casein phosphopeptides, we did not detect any milk-derived peptides with proposed bioactivity. The absorption behavior of two resistant ?-Lactoglobulin (?-Lg) domains, ?- Lg 125-135 and ?-Lg 40-60, was studied in detail using synthetic peptides. The IgE-binding properties of the peptide digests and synthetic peptides recovered from both the apical and basolateral compartments of the monolayers were evaluated by a dot-immunobinding assay using the sera of children (N=5) diagnosed with cow's milk allergy. Outcomes indicated that ?-Lg 127- 135, which is a Caco-2 processed form of ?-Lg 125-135, is a possible "immune sensitizing factors" in vivo. The almost complete loss of the IgE-binding affinity of both CN and WP after enzymatic digestion and transepithelial transport also points out the need of designing in vivo experiments to track the metabolic fate of dietary proteins.

7 páginas, 6 figuras., Soybean (Glycine max) 7S β-conglycinin is a seed storage protein consisting of homo- and hetero-trimers of three subunits, namely α (~67kDa), α' (~71kDa), and β (~50kDa), non-covalently associated. The N-glycans released from the whole β-conglycinin have been already characterized by 1H NMR some decades ago. Nevertheless, the actual glycosylation of the potential sites and the glycoforms of the individual subunits have not been specifically investigated so far. In this study, up-to-date chromatographic, electrophoretic and mass spectrometric strategies have been combined to achieve the structural characterization of the glycoforms of the three individual β-conglycinin subunits. Glycosylation sites were assigned by analyzing the tryptic glycopeptides of the isolated subunits. Underivatized N-glycans were purified with a two-step clean-up, consisting in sequential reversed-phase and activated porous graphitized carbon micro-chromatography, and profiled by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). © 2013 Elsevier B.V., The present research has been supported by the projects Italy-Spain Integrate Action (IT2009-0087, IT10ESiFCF2) and CONSOLIDER Ingenio 2010 (FUN-C-FOOD): CSD 2007-00063). Dr. M.A.-B. thanks CSIC and European Social Funds for her JAEdoc contract.

The carbohydrate moiety of human α-fetoprotein, an RMM 67000 glycoprotein produced in a hepatoma cell line (Hep G2), was investigated by the combined use of high-resolution chromatographic techniques and mass spectrometry. Fast atom bombardment mass spectrometric (FABMS) and reversed-phase high-performance liquid chromatographic analysis of α-fetoprotein obtained from a large-scale cell culture following tryptic and peptide N-glycanase F hydrolysis demonstrated that the protein contains a single glycosylation site at level of asparagine 232. Further, electrospray mass spectrometric measurement of the intact protein molecular mass showed that two main glycoforms are present. The complete definition of the structural heterogeneity of the oligosaccharide moiety was achieved by high-performance anion-exchange chromatography with pulsed amperometric detection together with carbohydrate mapping by FABMS of the released oligosaccharides demonstrating (i) that the glycosylation produced by cell culture is of the biantennary complex type typical of human hepatoma α-fetoprotein and (ii) the presence of two main structures in a ratio of about 2:1 differing in the presence of a fucose residue in the N-acetylglucosamine in the non reducing portion of the molecule.

SUMMARYThe heterogeneity of ovine β-casein, apparent after discontinuous gel electrophoresis at alkaline pH and gel isoelectric focusing either by staining with Coomassie blue or immunoblotting, was investigated in detail. Electrospray mass spectrometry was used to characterize ovine casein by direct examination of whole casein samples. β-Casein in individual milks consisted of several components which were characterized through the accurate determination of their molecular masses. It was demonstrated that protein species occurring simultaneously in individual ovine milks differed mainly in the number of phosphate groups.

The possibilities of characterizing the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) by liquid chromatography electrospray mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption with time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated. The B subunit from recombinant E. coli (expression in Pichia pastoris) can be detected by LC/ESI-MS expressed in P. pastoris and the charge envelope signals can be observed; LC/ESI-MS and MALDI-TOF-MS analysis allowed the acquisition of labile toxin subunit B (LTB) molecular weight and preliminary structural characterization of LTB toxin. MALDI-TOF analysis after reduction and alkylation of the protein evidenced the presence of one disulfide bond in the structure of the protein. Confirmatory analysis was carried out by detection of most of the tryptic fragments of the B subunit by MALDI-TOF-MS, obtaining total coverage of the protein sequence. Possible biovariations in the toxin can mostly be determined by sequencing, where an increase of molecular mass in the N-terminal side of the protein was identified. This modification may be due to an O-GlcNAc-1-phosphorylation.

Sakacin A was purified to homogeneity through simple chromatographic procedures from cultures of Lactobacillus sakei DSMZ 6333 grown on a low-cost medium. The highly purified protein dissipated both transmembrane potential (ΔΨ) and transmembrane pH gradient (ΔpH) in Listeria cells in a very intense, rapid, and energy-dependent fashion. On a slower timescale, purified sakacin A also showed a lytic activity toward isolated cell walls of Listeria. Mass spectrometry was used to analyze the products of sakacin A action on cell walls, evidencing that sakacin A acts on various types of bonds within peptoglycans.

SummaryThe non-protein nitrogen (NPN) of samples of Parmigiano-Reggiano cheese ripened for 6 and 15 months was fractionated by ion-exchange chromatography on a Cu2+-Chelex column to separate oligopeptides from free amino acids. Peptide components were isolated by reversed-phase HPLC and identified by fast atom bombardment–mass spectrometry (FAB–MS). Only the NPN fraction of 6 month old cheese samples contained enough peptides to be further characterized. On the basis of FAB–MS spectral results, 39 oligopeptides were identified, the main components being phosphopeptides. Two sets of both intact and partly dephosphorylated peptides, accounting for a total of 19 phosphopeptides, were formed by the hydrolysis of β–casein and belonged to regions 1–20 and 6–28 of β–casein. The formation and potential role of these peptides in cheese is discussed.

The cord blood sample of a Caucasian newborn contained about 40% of an abnormal fetal hemoglobin. The mutated γ chain was isolated using reversed phase high performance liquid chromatography and characterized by means of electrospray and fast atom bombardment mass spectrometric techniques as a Gγ-globin variant with an Ile->Thr substitution at position -γ75. The variant chain shows the same structure as the previously described Hb F-Charlotte that was demonstrated to be an Aγ variant with an Ile->Thr substitution at position γ75 and an additional Ala->Gly substitution at γ136.

In the last years proteomic science has started to provide an important contribution to the disclosure of basic aspects of food-related diseases. Among these, the identification of proteins involved in food allergy and their mechanism of activation of toxicity. Elucidation of these key issues requires the integration of clinical, immunological, genomic and proteomic approaches. These combined research efforts are aimed to obtain structural and functional information to assist the development of novel, more reliable and powerful diagnostic protocols alternative to the currently available procedures, mainly based on food challenge tests. Another crucial aspect related to food allergy is the need for methods to detect trace amounts of allergenic proteins in foods. Mass spectrometry is the only non-immunological method for high-specificity and high-sensitivity detection of allergens in foods. Nowadays, once provided the appropriate sample handling and the correct operative conditions, qualitative and quantitative determination of allergens in foods and ingredients can be efficiently obtained by MALDI-TOF-MS and LC–MS/MS methods, with limits of detection and quantification in the low-ppb range. The availability of accurate and fast alternatives to immunological ELISA tests may also enable the development of novel therapeutic strategies and food processing technologies to aid patients with food allergy or intolerance, and to support allergen labelling and certification processes, all issues where the role of proteomic science is emerging.

The in-depth characterization of water buffalo (WB) whey proteins based on chromatographic and mass spectrometric techniques revealed unexpected structural co- and post-translational modifications for β-lactoglobulin (β-Lg). The residues Lys⁴⁷ and Lys⁶⁹ of β-Lg were found to be lactosylated early, at the time of milking. Thiol groups of β-Lg underwent a dynamic sulfhydryl/disulfide exchange that is probably essential in accomplishing specific physiological requirements in which proteins may alternatively act either as a trigger or as a target. In this sense, the free sulfhydryl group of β-Lg established a glutathionylation/deglutathionylation equilibrium, which could be functional in conveying and delivering glutathione. Furthermore, the N-lauroylated β-Lg occurring exclusively in WB milk has been characterized for the first time. N-acylation could be an evolutionary remnant of ancestral lipocalins. Combined with the known aptitude of β-Lg to interact with phospholipid bilayers, this suggests that the protein could also be involved in the membrane translocation of small molecules, in addition to targeting, trafficking or the maintenance of membrane integrity. This structural characterization of β-Lg adds to the currently existing data and expands our understanding of the possible biological roles of this enigmatic protein.

1. 1. The globin chain components of Sprague-Dawley rat hemoglobin were obtained by reverse-phase HPLC which showed the presence of two α-chain and four β-chains. 2. 2. The accurate molecular weight of each globin chain was determined by means of electrospray mass spectrometry. Extensive mass spectrometric analysis on several enzymatic digests by fast atom bombardment mass spectrometry (FAB-overlapping) meant to determine the complete sequence of the α-major and of the four β-globins. 3. 3. The primary structure of the α-major globin was found in agreement with literature data (Garrick et al., 1975 Biochem. J.149, 245–258; Chua et al., 1987). 4. 4. Sequence analysis of the four β -globin chains showed that amino acid differences are restricted to two protein portions: the region 22–25 and 123–125, the remaining portions of the molecule being unchanged in the four globins. Furthermore, all the amino acid replacements correspond to single point DNA mutations and (with the exception of the substitution Asp 22 → Asn in the β2-globin) involve uncharged substitutions.

At present, compared with bovine milk, the characterization of donkey milk caseins is at a relatively early stage progress, and only limited data are related to its genetic polymorphism. In this work, the heterogeneity of donkey caseome was investigated using a proteomic approach, based on one- (PAGE, UTLIEF) and two-dimensional (PAGE-->UTLIEF) electrophoresis, stained with either Coomassie Brilliant Blue or specific polyclonal antibodies, and structural MS analysis. These combined methodologies allowed the contemporary identification of donkey alpha(s1), alpha(s2), beta and kappa-CN with their related heterogeneity due to phosphorylation (alpha(s1), alpha(s2) and beta-CN), glycosylation (kappa-CN) and incorrect splicing of RNA in mRNA (deleted forms of alpha(s1)-CN and beta-CN). The results achieved showed 11 components for kappa-CN, six phosphorylated components for beta and alpha(s1)-CN and three main phosphorylated components for alpha(s2)-CN, each accounting for 10, 11 and 12 P/mole. At this regard, for the first time, the primary structure of the expressed protein corresponding to the only available donkey alpha(s2)-CN cDNA sequence was determined. Furthermore beta-CN was found in homozygous and heterozygous state for the occurrence of a genetic beta-CN variant having a MW value 28 mass units higher than the common beta-CN phenotype.

Resistance to proteases throughout the gastrointestinal (GI) tract is a prerequisite for milk-derived peptides to exert biological activities. In this work an in vitro multi-step static model to simulate complete digestion of the bovine milk proteins has been developed. The experimental set-up involved the sequential use of: (i) pepsin, (ii) pancreatic proteases, and (iii) extracts of human intestinal brush border membranes, in simulated gastric, duodenal and jejuneal environments, respectively. Enzymatic concentrations and reaction times were selected in order to closely reproduce the in vivo conditions. The aim was to identify the peptide candidates able to exhibit significant bioactive effects. Casein and whey protein peptides which survived the in vitro GI digestion have been identified by the combined application of HPLC and mass spectrometry techniques. While the permanence of the main potentially bioactive peptides from both casein and whey proteins was found of limited physiological relevance, the high resistance to proteolysis of specific regions of beta-lactoglobulin (beta-Lg), and especially that of the peptide beta-Lg f125-135, could have implications for the immunogenic action of beta-Lg in the insurgence of cow's milk allergy.

The most challenging analytical task facing phosphoproteome determination requires the isolation of phosphorylated peptides from the myriad of unphosphorylated species. In the past, several strategies for phosphopeptide isolation have been proposed in combination with subsequent mass spectrometric investigations. Among these techniques, immobilized metal affinity chromatography and titanium dioxide have been recognized as the most effective. Here, we present an alternative method for the enrichment of phosphopeptides based on hydroxyapatite (HAP) chromatography. By taking advantage of the strong interaction of HAP with phosphate and calcium ions, we developed an efficient method for the selective separation and fractionation of phosphorylated peptides. The effectiveness and efficiency of recovery for this procedure was assayed using tryptic digests of standard phosphorylated protein mixtures. Based on the higher affinity of multi-phosphorylated peptides for HAP surfaces, the introduction of a phosphate buffer gradient for stepwise peptide elution resulted in the separation of mono-, di-, tri-, and multi-phosphorylated peptides. Thus, we demonstrated that this technique is highly selective and independent of the degree of peptide phosphorylation.

Proteomic techniques were applied for the investigation of “avenin-like protein type-B” in tetraploid wheat. Protein was extracted from endosperm, according to a classical chromatographic procedure and its identity confirmed by liquid chromatography-tandem mass spectrometry analysis. The characterization of avenin-like protein type-B was in good agreement with the gene-derived sequence, with the exception of glutamine as N-terminus. The presence in the glutenin extract and the high number of cysteine residues suggested that avenin-like protein type-B is integrated into gluten polymers via inter-chain disulphide bonds. This raises the question of whether the protein could play a role in determining the functional properties of gluten.

SummaryThe isolation and identification of low molecular mass peptides formed during the ripening of Parmigiano-Reggiano cheese is described. A strategy was used based on the fractionation of nitrogenous material using chemical methods followed by HPLC to isolate peptides and fast atom bombardment-mass spectrometry to identify them. It was found that the majority of cheese oligopeptides arose from the proteolysis of β-casein. Several phosphopeptides and oligopeptides known in vivo to be biologically active have also been identified during the ripening of cheese.

Cylindrospermopsin (CYN) is a toxic alkaloid-like compound produced by some strains of cyanobacteria, procariotic organisms occurring in water blooms, observed worldwide in eutrophic lakes and drinking water reservoirs. Methods for determination of CYN in freshwater and fish muscle by liquid chromatography coupled to electrospray ion trap mass spectrometry are herein described. The performances of both methods are reported; ion trap LC/ESI-MS/MS resulted highly selective and reliable in unambiguous identification of CYN, based on monitoring the precursor ion and three product ions. The methods developed showed satisfactory mean recoveries (higher than 63.6%) and relative standard deviations, ranging from 5.8 to 9.8%. The limits of quantification at 0.10 ng/mL in freshwaters and 1.0 ng/g in fish muscle, respectively, allow for determination of CYN also in early contamination stages. Ion trap LC/ESI-MS/MS was successfully applied to the identification and quantification of CYN in water and cyanobacteria extracts from Lake Averno, near Naples, representing the first case of contamination described in Southern Italy.

Cyanobacteria (blue-green algae) cause blooms in eutrophic lakes and drinking water reservoirs. They also produce biotoxins, including microcystins (MCs), highly toxic cyclic heptapeptides that cause poisoning in animals and human. In this paper, we present a method for the analysis of four MCs by ion trap LC-MS and MALDI-TOF/MS. The data are compared to evaluate the performance and reliability of the different MS detection systems. The method was applied to the analysis of water and algae samples from Lake Averno, near Naples, as a consequence of a cyanobacteria bloom. The analysis of algae cell extracts showed no contamination by known microcystins, but the three main substances were detected. MALDI-TOF/MS was successful for screening of the biotoxins in the samples, identifying anabaenopeptin B and anabaenopeptin F as the major contaminants on the basis of literature mass spectrometry data. The structure of the third compound was not identified and is under further investigation. The method could characterise the biotoxins produced in Lake Averno for evaluating health risks related to their presence.

Mass spectrometry has arguably become the core technology for the characterization of food proteins and peptides. The application of mass spectrometry-based techniques for the qualitative and quantitative analysis of the complex protein mixtures contained in most food preparations is playing a decisive role in the understanding of their nature, structure, functional properties and impact on human health. The application of mass spectrometry to protein analysis has been revolutionized in the recent years by the development of soft ionization techniques such as electrospray ionization and matrix assisted laser desorption/ionization, and by the introduction of multi-stage and ‘hybrid’ analyzers able to generate de novo amino acid sequence information. The interfacing of mass spectrometry with protein databases has resulted in entirely new possibilities of protein characterization, including the high sensitivity mapping (femtomole to attomole levels) of post-translational and other chemical modifications, protein conformations and protein–protein and protein–ligand interactions, and in general for proteomic studies, building up the core platform of modern proteomic science. MS-based strategies to food and nutrition proteomics are now capable to address a wide range of analytical questions which include issues related to food quality and safety, certification and traceability of (typical) products, and to the definition of the structure/function relationship of food proteins and peptides. These different aspects are necessarily interconnected and can be effectively understood and elucidated only by use of integrated, up-to-date analytical approaches. In this review, the main aspects of current and perspective applications of mass spectrometry and proteomic technologies to the structural characterization of food proteins are presented, with focus on issues related to their detection, identification, and quantification, relevant for their biochemical, technological and toxicological aspects.

Cyanobacteria, also called blue-green algae, occur worldwide within water blooms in eutrophic lakes and drinking water reservoirs, producing several biotoxins (cyanotoxins). Among these, microcystins (MCs) are a group of cyclic heptapeptides showing potent hepatotoxicity and activity as tumour promoters. So far, at least 89 MCs from different cyanobacteria genera have been characterised. Herein, ion trap, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) and quadruple time-of-flight (Q-ToF) mass spectrometry (MS)-based methods were tested and compared for analysing MCs in freshwaters. Method performances in terms of limit of detection, limit of quantification, mean recoveries, repeatability, and specificity were evaluated. In particular, a liquid chromatography/electrospray ionisation (LC/ESI)-Q-ToF-MS/MS method was firstly described to analyse MCs in freshwaters; this technique is highly selective and sensitive, and allowed us to characterise the molecular structure of an unknown compound. Indeed, the full structural characterisation of a novel microcystin variant from a bloom of Planktothrix rubescens in the Lake Averno, near Naples, was attained by the study of the fragmentation pattern. The new cyanotoxin was identified as the 9-acetyl-Adda variant of microcystin-RR.

Currently, the internally deleted caprine alphas1-casein (alphas1-CN) variants F and G, associated with low casein expression, are detected by means of ordinary descriptive techniques. No relevant procedure is available to detect internally deleted goat alphas1-CN in bulk milks. The availability of full-length and alphas1-CN F and G variants allowed us to further investigate this issue. Using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-MS and ESI-MS/MS, tryptic signature peptides alphas1-CN F f59-63/f43-63, alphas1-CN G f4-20/f4-21, alphas1-CN B2 f4-22 Pro16 and alphas1-CN A f4-22 Leu16 were identified. This also helped to solve the interesting question of how the casein variants contribute to the composition of goat's bulk milk. Synthetic peptide analogues with ionization efficiency equivalent to that of tryptic junction peptides were used as internal standards to evaluate alphas1-CN variants, either individually or globally, using bulk milk from a single goat breed as a model system. Here, alphas1-CN F accounted for 0.15+/-0.08% and the alphas1-CN G variant was missing or below the 0.10% detection limit. The analysis of six samples confirmed that alphas1-CN G was missing and that alphas1-CN F occurred at a low frequency in hybrid and local breed bulk milks from Mediterranean areas. In conclusion, a quantitative MS-based application of the signature peptides for full-length and internally deleted variants in goat's casein is provided. The strategy is also suggested for the determination of splice variants in any biological sample.

The unnatural amino acid lysinoalanine (LAL) has been identified in milk and cheese products by liquid chromatography mass spectrometry (LC/ESI/MS) with selective ion monitoring (SIM) of the 9-fluorenyl-methylchloro-formate (FMOC) derivative. LAL is not present in raw milk or derived from Mozzarella cheese; however, high amounts of LAL are found in calcium caseinate and milk powder. As expected, milk fortified with caseinate or whey protein powder produces cheese with higher LAL content. Our analytical procedure is based on the simultaneous detection of specific ion masses of the FMOC–LAL derivative and the N - e -methyl-lysine internal standard. A linear relationship was observed within the 0.2–20 ppm concentration range, in addition to a high correlation coefficient and ∼3% relative standard deviation.

One of the main objectives of wheat glutenin subunit (GS) analysis is the identification of protein components linked to wheat quality. The proteomic characterization of glutenin has to consider the relatively low levels of arginine and lysine residues and the close sequence similarity among the different groups of these subunits, which hinders or even prevents the identification of the GS. In this study, a proteomic approach has been applied to resolve the heterogeneity of wheat glutenin components. Proteins extracted from Triticum durum flour were first analyzed by two-dimensional gel electrophoresis, which greatly reduced glutenin complexity. The identity of each spot was confirmed by nano liquid chromatography tandem mass spectrometry analysis of tryptic peptides. In parallel, measurements of the high mass range by matrix-assisted laser desorption/ionization time-of-flight analysis allowed detection of the large tryptic peptides. Gathering all data from search engine interrogation, very high sequence coverage was obtained for high molecular weight GS, including Bx7 and By8, in agreement with the known genetic profile of durum wheat. In addition, a truncated form of By8, never detected before, was also found. Low molecular weight GS (LMW-GS) B-type was identified with reasonable sequence coverage, while a clear identification of LWM-GS C- and D-type was hindered by the incompleteness of the wheat DNA databases. This study represents the first comprehensive analysis of the glutenin proteome and provides a reliable method for classifying wheat varieties according to their glutenin profile.

Current knowledge indicates that both innate and adaptive immune responses are involved in Celiac disease (CD) driven by different gliadin peptides. By studying a representative recombinant α-gliadin form, a further 25-mer peptide resistant to gastric, pancreatic, and human intestinal brush-border membrane enzymes was detected. This peptide latter encompasses the sequence 31–43 known to elicit the innate immune response in CD. The resistance of 25-mer, as well as that of the already described 33-mer related to the CD adaptive immune response, was confirmed on a standard flour wheat sample representative of the most widespread European varieties.

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function remains unclear. A complete definition of the physiological activities of this oncofetal protein has been severely limited, until now, by the lack of a purification procedure appropriate to obtain pure AFP in appreciable amount. The present report describes a purification procedure extremely rapid and simple and takes advantage of the well-known fact that AFP contains copper. We have developed a single-step purification procedure by immobilized copper-chelate affinity chromatography using the culture medium from human hepatoblastoma cell line HepG2 grown in the absence of serum. This method yields AFP at high purity and high yield. Purified AFP amino acid sequence, molecular mass, carbohydrate structure, and copper content were found to be in line with previous studies. Moreover, we found that the purified AFP has superoxide dismutase activity with efficiency similar to that of the native Cu, Zn SODs at physiological pH. This result may provide further support to the idea that AFP is a bifunctional protein, acting in cellular defence against oxidative stress both as a copper buffer and as a superoxide radical scavenger.

In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate comparison of 2D maps to define the events occurring during their ripening and individuate candidate molecular markers of the characteristic proteolytic processes. Considering that, essentially, muscle endogenous enzymic activity, calpains and cathepsins, occur in the ripening process of dry-cured ham, whereas a combined action between endogenous and microbial enzymes takes place in the case of sausage ripening, these results provide deeper insight into the respective role of endogenous and microbial enzymes in performing proteolysis. Finally, image analysis and creation of data bank could be achieved to quickly identify and protect typical products.

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.