Food allergies are a serious health concern with increasing worldwide prevalence. Food legislation issued in several countries requires detailed declarations of allergenic ingredients in food products; however, an important risk for allergic consumers is posed by the presence of undeclared allergens due to potential cross-contaminations during food processing. Sensitive, reliable and robust methods are necessary to trace food allergens and reduce the risk of accidental exposure. Detection methods may target the allergen itself, or a marker for the allergen presence (either protein or DNA), the final choice strictly depending on the information available for the target allergen and the specific antibodies and primers. Current common methods for food allergen analysis utilize antibody-based assays although some drawbacks can be encountered such as matrix/processing effects and epitope masking especially when dealing with complex and processed foods. Alternative non-immunological methods have been investigated in the last decade with particular emphasis placed on mass spectrometry based method.In the present contribution, an overview of the main methodological approaches will be presented critically discussing the main pro and cons of each approach.

The risks associated with the presence of hidden allergens in the food chain have raised the need for fast, sensitive, and reliable methods to trace food allergens in different commodities. We highlight advances and future trends in biosensor systems applied to food-allergen management. We discuss critical aspects of biosensor development with particular emphasis on integrating nanotechnology.

Food allergy (FA) is a relatively new food safety issue still deemed to be on the rise in the recent years and with an increasing number of food allergens identified and a growing number of consumers suffering from FA. No actual cure currently exists for food allergy and therefore food allergic consumers can only manage their condition by carefully avoiding products which contain the allergenic food(s). In order to address this effectively, food allergic consumers absolutely rely on the availability, accuracy and reliability of information provided on foods they intend to buy, resulting in the urgent need for specific labeling legislation. On this regard, the European Union (EU) is very restrictive, with the last Directive 2007/68/EC requiring the mandatory labeling of a total of 14 allergenic ingredients, whenever used and irrespective of the amounts, on the respective food label with few exemption cases also listed in the same Directive [1]. This regulatory framework is intended to provide consumers with information about allergens when they are introduced in foods as ingredients; however, allergens may also inadvertently contaminate the food when manufacturing processes and control measures are not adequate to prevent cross-contact between allergen-containing and allergen-free foods then becoming "hidden allergens". This last represents the major threat for allergic consumers and for food manufacturers, the latter being responsible for the safety of food products brought onto the market.In the last two decades, mass spectrometry has played a pivotal role in proteomic research being the election method for protein identification in complex mixtures. Such MS approach has been implemented in the food allergen field proving to be a reliable confirmatory tool overcoming the objective limitations of antibody-based kits commercially available, accounted by epitope masking phenomena or epitope modification occurring upon application of thermal treatments [2]. Additional advantages rely on the possibility to run multiple-allergen analysis in one shot, quantitative analysis, structural protein elucidation, characterization of protein modifications and epitope mapping. In the last five year, our Institute actively contributed to such research field thanks to the acquisition of last-generation high resolution mass spectrometers (HR-MS) based on OrbitrapTM technology with very high selectivity and sensitivity achieved in the food allergen detection methods developed [3-5].In the present communication, different examples of our achievements will be presented and critically discussed, highlighting the relevance and benefit of the accurate mass detection of allergens markers for the fulfillment of specific objectives. Features and analytical performances of different HR-MS platforms were investigated and will be thoroughly discussed along with their strengths and weaknesses applied to the multi-allergen screening in different food matrices.

Bio-sensing represents one of the most attractive applications of carbon material based electronic devices; nevertheless, the complete integration of bioactive transducing elements still represents a major challenge, particularly in terms of preserving biological function and specificity while maintaining the sensor's electronic performance. This review highlights recent advances in the realization of field-effect transistor (FET) based sensors that comprise a bio-receptor within the FET channel. A birds-eye view will be provided of the most promising classes of active layers as well as different device architectures and methods of fabrication. Finally, strategies for interfacing bio-components with organic or carbon nano-structured electronic active layers are reported.

Owing to its extensive use in human diet, wheat is among the most common causes of food-related allergies and intolerances. Gluten proteins and particularly the gliadin fractions represent the main factor triggering celiac disease. Given the extremely high structural heterogeneity of gliadins, generated by amino acid insertions, deletions and substitutions, the physico-chemical properties of gliadins can vary significantly among wheat genotypes (species, cultivars and breeding lines) influencing in parallel the immunoreactive properties and the susceptibility to enzymatic treatment [1]. Therefore, the structural characterization and the correlation with relevant toxicity, by tracking the fate upon gastrointestinal digestion of wheat-based commodities [2], gains significance to deepen the knowledge at the molecular level of the immunological pathway and to identify naturally low toxic wheat species and/or efficient detoxification technologies.Recent development in proteomics have contributed to give insights in this field, although the analytical capabilities of the proteomic approach are challenged by the complexity of the wheat seed proteome and particularly of the gluten protein fraction. Limited database entries available, complexity arising from sets of homologue proteins, large occurrence of repeated motifs, very low number of basic residues for tryptic hydrolysis represent drawbacks that complicate the comprehensive proteomic cataloguing of the gluten proteins. These challenging issues can only be addressed by the use of integrated, up-to-date analytical approaches, which together constitute the platform of modern food proteomics, and where a pivotal role is played by mass spectrometry.

The improvement of a surface plasmon resonance (SPR)-based immunoassay for the detection of traces of egg-based fining agents in red wines is herein described. The latter represents an extension of a previously developed direct assay targeted to the detection of ovalbumin (OVA) as marker of the presence of egg white powder residues, a typical fining agent utilized by the winery industry. In this paper, a suitable pre-treatment procedure was optimized for the sensitive detection of OVA at sub-ppm levels in red wines fortified with egg-white powder, by using an immunoassay proved to be reliable for both white and roseé wines. A red wine from Chianti grapes selected as reference matrix was artificially contaminated with egg-white powder before undergoing different sample purification. Several purification strategies were investigated and tested in order to challenge the limit of detection (LOD) obtained with the methods currently in use for egg allergen detection. Finally, the optimized two-step pre-treatment, combining polyvinylpolypyrrolidone-based purification and size exclusion chromatography, enabled to achieve an LOD in red wine as low as 0.2 ?g/mL. The optimized SPR-based method met the method performance criteria issued by the International Organization of Vine and Wine (OIV) concerning the minimum sensitivity required for the analyses of potentially allergenic fining agent proteins in wines, confirming the biosensor as promising tool to monitor the residual contamination level of fined red wines.

The identification of wheat genotypes with low toxicity could represent a valid alternative for the prevention of wheat intolerance onset. Over the last years, great efforts have been devoted to develop effective gluten detoxification strategies mostly based on enzymatic strategies, which, however, involve a simultaneous detrimental alteration of the technological properties. In this frame, obtaining low-gluten wheat products without affecting their rheological properties is still a challenging issue.In this contribution, we presented an integrated approach encompassing both proteomic characterization and grains yield/quality evaluation for the identification of durum wheat genotypes combining potential lower toxicity/immunogenicity with satisfactory rheological properties. A preliminary profiling of gluten proteins was accomplished by immunoassay-based quantification and liquid chromatography coupled to UV detection focusing on the gliadin fraction as main responsible for immunoreactivity in celiac disease patients. In addition, complementary information about productivity-related traits and quali-quantitative characteristics were collected, such as grain protein content, grain yield per spike, dry gluten and gluten index. The whole pool of data was statistically evaluated confirming that durum wheat breeding programs accomplished in the last 25 years improved the pasta-making quality (gluten strength), without causing an increment of toxic epitopes towards CD patients. Tracking the fate of gluten proteins upon in-vitro simulated gastroduodenal digestion experiments and in-silico assessing the risk of toxicity according to the most recent guidance provided by EFSA1, confirmed such statement. The selected genotypes boasting medium and strong gluten strength, all presented a significantly lower number of toxic epitopes compared to commercial semolina. In perspective, such genotypes could represent an innovative alternative for preventive and therapeutic wheat based foods in genetically predisposed individuals who may develop CD after prolonged wheat or gluten consumption.

Food allergies are a serious health concern with increasing worldwide prevalence. Food legislation in place in several countries, requires detailed declaration of allergens in foods implying capability of methodologies to reliably trace food allergens. However, detecting and quantifying food allergens remains a challenge. Current common methods for food allergen analysis utilize antibody-based assays although some drawbacks are encountered such as matrix/processing effects and epitope masking especially when dealing with complex and processed foods. Therefore, sensitive, reliable, robust, fast, reproducible, and standardized methods are necessary for improved allergen analysis and reduce the risk of allergen contamination. In the last decade, mass spectrometry (MS) techniques have been developed and applied with success to food allergen detection. This review compares different aspects of food allergen quantification using advanced MS techniques including multiple reaction monitoring. The latter provides low limits of quantification for multiple allergens in complex food matrices, while being robust and reproducible. (C) 2018 Elsevier B.V. All rights reserved.

Mass spectrometry (MS) represents an essential tool in proteomics studies, in the last years also exploited for monitoring allergens contamination in food products. Milk and egg are renowned allergens often used as fining agents to promote clarification of wines, therefore any residual amount in the endproducts could represent a menace for allergic individuals. In view of this, it is of paramount importance to have at disposal sensitive analytical methods able to detect traces of milk and egg allergens in food. In this work we describe the upgrade and the optimization of an analytical workflow based on the use of a pre-enrichment column coupled with HPLC separation and MS/MS detection for the selective and sensitive detection of milk and egg allergens in white wine. Two different sample pre-treatments based on the use of mass cut-off filters or size exclusion cartridges were evaluated and compared, before tryptic digestion and LC-SRM-MS/MS analysis of the resulting peptides mixture. The devised UF based method coupled with peptide on-line pre-enrichment enabled to reach the lowest LODs down at 0.036 ug/mL and 0.05 ug/mL for egg and milk allergens respectively, proving to be the most sensitive strategy for monitoring allergens contamination in wine.

The present work is aimed at developing gold nanostructures functionalized with antenna systems to exploit the synergistic nanostructure/antenna desorption-ionization efficiency. A potential Matrix-Assisted Laser Desorption Ionisation (MALDI) organic matrix has been modified introducing specific functional groups or molecular linker and used as a capping agent for gold nanostructures. In particular, conjugated naphthyl-thio-derivative, i.e. 4-mercaptonaphthalene-1,8-dicarboxylic acid, was synthesized and characterized by means of nuclear magnetic resonance, UV-visible and X-ray photoelectron spectroscopies. Afterwards, the thio-derivative was used as covalent surface modifier for flat gold surfaces and nanostructured gold films. These surfaces were thoroughly characterized by means of parallel angle-resolved X-ray photoelectron spectroscopy to obtain quantitative information about elemental composition, chemical speciation, and in-depth distribution of the target chemical functional groups. Finally the compound was preliminarily tested as a non-conventional matrix in Laser Desorption Ionisation Mass Spectrometry (LDI-MS) analysis of low molecular weight biomolecules in order to assess its capability of acting as the antenna system and proton donor after covalent bonding to gold nanomaterials. © 2013 Elsevier Ltd. All rights reserved.

A Mass Spectrometry ImmunoAssay (MSIA) specifically designed for the detection of egg allergens in wines is described. This approach is based on an immunoaffinity enrichment procedure combined with targeted MS/MS detection of selected egg peptide markers. Polyclonal antibodies raised against native ovalbumin, chosen as the target protein tracing for egg powder, were immobilized onto low backpressure monolithic MSIA customized disposable tips. Ovalbumin-free wine samples were fortified with standard protein at different concentration levels in the low µg/mL range. A simple protocol was devised consisting in a 1:4 dilution of the wine sample with a basic solution for pH adjustment, followed by a semi-automated purification/enrichment step on customized MSIA disposable tips fitted on a multichannel electronic pipette. Among the main figures of merit LOD and LOQ values as low as 0.01 and 0.03 ?g/mL, respectively, of 18 % should be noticed. Noteworthy, the developed assay outperformed current MS-based methods for the detection of allergenic protein in wine matrices, thanks to the immunoaffinity enrichment. In addition compared to other immunoassays, the present approach boasts the unquestionable advantage to provide an unambiguous identification of the target protein, by simultaneous detection of three unique peptide markers each giving three specific MS/MS transitions.

Food allergy is nowadays regarded as a problem of public-health relevance, the main concern being the unintentional exposure of allergic consumers to the offending ingredient through allergen-containing food. Rapid diagnostic tools are increasingly being requested by food companies to verify the efficiency of their management schemes for food safety. Although no specific reference analytical method for the determination of fining agent proteins has been prescribed, the international Organization of Vine and Wine (OIV) resolution 427-2010 modified by the OIV/COMEX 502-2012 set up the analytical requirements to be fulfilled by methods under development. In particular, ELISA methods must comply with the detection limits and the quantification limits of <=0.25 and 0.5 mg/L, respectively.In the present communication, the development of a surface plasmon resonance (SPR)-based biosensor tailored to the fast detection of egg related fining allergens in wines is described. Ovalbumin (OVA) was chosen as target protein to be monitored due to its highest abundance in the egg white (EW) powder, a typical fining agent used by the winery industry to promote wine clarification. A direct assay was designed, basing on the use of polyclonal anti-OVA antibody as bio-specific receptor. After the fine tuning of all parameters able to influence the final response, the assay was tested in a direct assay for OVA in commercial wines artificially contaminated with EW powder. The devised assay allowed to trace, in a short analysis time and with a minimal sample pre-treatment, the presence of egg allergens at the lowest concentration comprised between 0.03 and 0.2 ?g/mL [1].

Recent technological advances in instrumentation and sample preparation have raised the attention towards MS-based targeted protein immunoassays as attractive alternative to classical ELISAs [1]. The Mass Spectrometry ImmunoAssay (MSIA(TM)) approach utilizes the MSIA D.A.R.T.'s technology which comprises a molecular trapping micro-column contained within a pipette housing. A capture antibody is immobilized onto the micro-column and the antigen containing sample is applied by repetitive pipetting cycles. After washing and elution steps, the target antigen can be either directly injected into mass spectrometers or subjected to enzymatic digestion for peptide markers detection. The combination of, immunoaffinity capture and mass spectrometry provide complementary advantages: the first enables a specific binding of the target molecule from a complex matrix using the immobilized antibody with its consequent selective enrichment on the micro-column and the prospective to fully automate the procedure; MS instead provides an unambiguous detection, as antigen signals are observed at characteristic m/z values in the mass spectrum, overcoming common issues of immunoassays, such as false positives.So far, MSIA technology has found mainly applications in clinical fields where the complexity of biofluids, such as blood plasma and serum, impairs the sensitivity, robustness and throughput of routine MS-based protein detection [2 and reference thereof].In the present investigation, MSIA approach was applied to the food safety field. Notably, the applicability of a MSIA based workflow was tested for the detection of residual egg proteins in wine. Egg-derived products, in various commercial preparations, are commonly utilized in winemaking thanks to the ability to interact and promote precipitation of wine polyphenols and other undesirable compounds [3]. However, any residues of egg white proteins remaining in wine could represent a risk for allergic consumers. In this scenario, the development of analytical approaches for the detection of egg proteins might open new perspectives for the producers, that might spot the real risk associated to certain procedures where allergens are likely to remain as residues. Till now, no official method for the determination of fining agent proteins is prescribed.Here, the feasibility of the MSIA approach for the detection of such residues in white wines was investigated, combining the semi-automated immunoaffinity enrichment with selective reaction monitoring (SRM) detection of specific peptide markers. The micro-columns in the MSIA D.A.R.T's were chemically modified to bind a polyclonal antibody commercially available to specifically recognize native ovalbumin (OVA), the most abundant protein in the egg based fining agent typically used for wine clarification. As a proof of concept, the MSIA D.A.R.T's were employed with the Finnpipette(TM) Novus i Multichannel Electronic Pipette which processes up to 12 samples simultane

Several buffer compositions were compared for their efficiency in protein extraction from both raw and roasted peanut and hazelnut samples, the final goal being to understand the modification of protein solubility upon roasting and maximize the extraction yield. Denaturant conditions provided by urea-TBS buffer resulted in satisfactory extraction yields for both peanut and hazelnut samples, before and after the thermal treatment. In addition, different varieties of peanuts and hazelnuts were characterized to highlight the extent of variability in the protein profile accounted by the varietal factor and eventual differential resistance among cultivars to protein modification induced by the thermal processing. The protein profile was characterized by gel electrophoresis, and specific bands were analyzed by micro-HPLC-MS/MS coupled to software-based protein identification. No significant difference was observed for the investigated hazelnut cultivars, namely, Campana, Romana, and Georgia, whereas interesting features were presented for the peanut varieties Virginia, Zambia, and China. In particular, Zambia variety lacked two bands of approximately 36 and 24 kDa that were visible in Virginia and China varieties, which could suggest a lower allergenic potential of this particular variety which deserves to be further investigated before drawing final conclusions.

In the present study, gold/surfactant core/shell colloidal nanoparticles with a controlled morphology and chemical composition have been obtained via the so-called sacrificial anode technique, carried out in galvanostatic mode. As synthesized Au-NPs had an average core diameter comprised between 4 and 8 nm, as a function of the electrochemical process experimental conditions. The UV-Vis characterization of gold nanocolloids showed clear spectroscopic size effects, affecting both the position and width of the nanoparticle surface plasmon resonance peak. The nanomaterial surface spectroscopic characterization showed the presence of two chemical states, namely nanostructured Au(0) (its abundance being higher than 90%) and Au(I). Au-NPs were then deposited on the top of a capacitive field effect sensor and subjected to a mild thermal annealing aiming at removing the excess of stabilizing surfactant molecules. Au-NP sensors were tested towards some gases found in automotive gas exhausts. The sensing device showed the largest response towards NOx, and much smaller - if any - responses towards interferent species such as NH3, H2, CO, and hydrocarbons

Nanomaterials have emerging importance in laser desorption ionization mass spectrometry (LDI-MS) with the ultimate objective being to overcome some of the most important limitations intrinsically related to the use of conventional organic matrices in matrix-assisted (MA) LDI-MS. This review provides a critical overview of the most recent literature on the use of gold nanomaterials as non-conventional desorption ionization promoters in LDI-MS, with particular emphasis on bioanalytical applications. Old seminal papers will also be discussed to provide a timeline of the most significant achievements in the field. Future prospects and research needs are also briefly discussed. © 2011 Springer-Verlag.

Almond is consumed worldwide and renowned as a valuable healthy food. Despite this, it is also a potent source of allergenic proteins that can trigger several mild to life-threatening immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus affecting the respective allergenicity. In this paper, we investigated the effect of autoclaving, preceded or not by a hydration step, on the biochemical and immunological properties of almond proteins. Any variation in the stability and immunoreactivity of almond proteins extracted from the treated materials were evaluated by total protein quantification, Enzyme Linked Immunosorbent Assay (ELISA), and protein profiling by electrophoresis-based separation (SDS-PAGE). The sole autoclaving applied was found to weakly affect almond protein stability, despite what was observed when hydration preceded autoclaving, which resulted in a loss of approximately 70% of total protein content compared to untreated samples, and a remarkable reduction of the final immunoreactivity. The final SDS-PAGE protein pattern recorded for hydrated and autoclaved almonds disclosed significant changes. In addition, the same samples were further submitted to human-simulated gastro-intestinal (GI) digestion to evaluate potential changes induced by these processing methods on allergen digestibility. Digestion products were identified by High Pressure Liquid Chromatography-High Resolution Tandem Mass Spectrometry (HPLC-HRMS/MS) analysis followed by software-based data mining, and complementary information was provided by analyzing the proteolytic fragments lower than 6 kDa in size. The autoclave-based treatment was found not to alter the allergen digestibility, whereas an increased susceptibility to proteolytic action of digestive enzymes was observed in almonds subjected to autoclaving of prehydrated almond kernels. Finally, the residual immunoreactivity of the GI-resistant peptides was in-silico investigated by bioinformatic tools. Results obtained confirm that by adopting both approaches, no epitopes associated with known allergens survived, thus demonstrating the potential effectiveness of these treatments to reduce almond allergenicity.

Mass spectrometry (MS) has played a pivotal role in proteomic research being the election method for protein identification in complex mixtures. In the last decade, the MS-based proteomic approaches have demonstrated to be a valuable confirmatory tool for allergen contamination management [1]. Our research group, actively contributed to such field developing label-free quantitative methods for the multiple allergen detection in several food matrices based on both low and high-resolution mass spectrometers [2-8]. Thanks to the advances provided by last-generation high resolution mass spectrometers (HR-MS) based on OrbitrapTM technology very high selectivity and sensitivity were achieved by the developed methods.In the present communication, performance provided by a hybrid quadrupole-OrbitrapTM MS platform will be presented. In particular, different acquisition modes were compared: Full-MS acquisition, targeted-Selected Ion Monitoring with data-dependent fragmentation (t-SIM/dd2) and Parallel Reaction Monitoring (PRM). The different acquisition modes were tested towards the detection of specific peptide markers arising from five different allergenic ingredients (milk, egg, soy, hazelnut, peanut) in home-made incurred cookies, selected as model processed matrix. In order to challenge the HR-MS platform, the sample pretreatment was kept as simply as possible, limited to a 30 min protein extraction followed by quick purification based on size exclusion chromatography by disposable cartridges. The three acquisition modes were independently optimized and compared in term of sensitivity, by means of ad-hoc calibration curves. In addition, performances provided by such hybrid HR-MS platform were compared with an optimized HPLC-ESI-SRM method we recently developed based on linear ion trap MS spectrometer [7] for the same kind of processed food matrix.

Peanut represents one of the most harmful allergenic foods capable of triggering severe and sometimes lethal reactions in allergic consumers upon ingestion of even small amounts. Several proteins capable of inducing allergic reactions that have been recognised by patients' IgE antibodies have been identified from this nut source. Methods mainly based on ELISA assays have been developed in order to detect peanuts in several food commodities. In addition LC-MS/MS methods based on different mass analysers have also been devised for tracing peanut contamination in different foods achieving low limits of detection. The applicability of a benchtop high-resolution Exactive(TM) mass spectrometer has never been investigated for the rapid screening of peanut contamination in complex food matrices like mixtures of nuts. We report in this paper the design of suitable peanut markers and the development of an high-resolution Orbitrap(TM) mass spectrometer-based method for peanut detection in a mixture of nuts species. With this aim, different types of samples were prepared: (1) nuts-based powder made up of a mixture of hazelnuts, pistachios, almonds and walnuts; and (2) nuts powder fortified with peanuts. Different levels of fortifications were produced and the applicability of the method was tested. Finally, a subset of six peptides fulfilling specific analytical requirements was chosen to check the suitability of the method tailored to the detection of peanuts in nuts-based products, and two of them, peptides VYD and WLG, were selected as quantitative markers. The method proved to be a suitable screening tool to assess the presence of traces of peanuts in other tree nuts with a limit of detection as low as 4 µg of peanuts proteins or 26 µg of peanuts in 1 g of matrix.

In recent years, mass spectrometry (MS) has been establishing its role in the development of analytical methods for multiple allergen detection, but most analyses are being carried out on low-resolution mass spectrometers such as triple quadrupole or ion traps. In this investigation, performance provided by a high resolution (HR) hybrid quadrupole-Orbitrap MS platform for the multiple allergens detection in processed food matrix is presented. In particular, three different acquisition modes were compared: full-MS, targeted-selected ion monitoring with data-dependent fragmentation (t-SIM/dd2), and parallel reaction monitoring. In order to challenge the HR-MS platform, the sample preparation was kept as simple as possible, limited to a 30-min ultrasound-aided protein extraction followed by clean-up with disposable size exclusion cartridges. Selected peptide markers tracing for five allergenic ingredients namely skim milk, whole egg, soy flour, ground hazelnut, and ground peanut were monitored in home-made cookies chosen as model processed matrix. Timed t-SIM/dd2 was found the best choice as a good compromise between sensitivity and accuracy, accomplishing the detection of 17 peptides originating from the five allergens in the same run. The optimized method was validated in-house through the evaluation of matrix and processing effects, recoveries, and precision. The selected quantitative markers for each allergenic ingredient provided quantification of 60-100mugingred/g allergenic ingredient/matrix in incurred cookies.

Soy is an important component of the human diet thanks to its nutritional value and high protein content; however, it also represents a risk for allergenic consumers due to its potential to trigger adverse reactions in sensitized individuals. The putative correlation between immunoreactivity and resistance to the human gastrointestinal (GI) digestion has drawn attention to investigating soybean proteins digestibility. In this work, we provided further insights into this field by performing in vitro simulated GI digestion experiments directly on ground soybean seeds, to provide more realistic results obtained from the digestion of the whole food matrix. Soybean digestion products were analyzed by SDS-PAGE followed by untargeted HPLC-MS/MS analysis and the final data were software treated to enable protein/peptide identification. The latter allowed monitoring the proteolytic degradation of the main soybean proteins during the gastric and duodenal phases. In particular, beta-conglycinin and trypsin inhibitors showed the highest resistance to the combined activity of GI enzymes, showing a partial degradation at the end of the duodenal phase as ascertained by the strong electrophoretic bands displayed at 50 kDa and 20 kDa, respectively. Glycinin subunits also presented, even if to a lower extent, resistance to the complete proteolytic degradation, as demonstrated by polypeptide fragments with molecular weight lower than 20 kDa displayed in the gel at the end of duodenal digestion. In addition, by bioinformatics analysis it was demonstrated that the GI resistant fragments of the allergenic proteins, beta-conglycinin and glycinin, retained in their primary structure linear epitopes potentially able to trigger an immunoreaction when exposed to the intestinal mucosa. Moreover, such resistant peptides also presented a structural homology with epitope sequences recognized in other legume species, presenting a potential risk of adverse cross-reaction for a larger category of allergic consumers.

IntroductionEffective enzymatic gluten detoxification strategies have been developed in the last years; however, obtaining low-gluten wheat products without impairing the rheological properties remains a challenging issue [1]. In this contribution, we presented an integrated analytical approach for the identification of wheat genotypes with reduced toxicity and satisfactory rheological properties [2].MethodsA comprehensive characterization of durum wheat genotypes was performed including grain quality traits (productivity-related and quali-quantitative characteristics) and proteomic profiling (R5-ELISA, HPLC-UV analysis). The data were evaluated statistically, and a selected list of candidates was subjected to in-vitro gastroduodenal digestion [3] and discovery MS/MS analysis for in-silico toxicity risk assessment [4].Results38 accessions of Triticum turgidum sp., including both wild and cultivated ones, were investigated. A preliminary profiling of gluten proteins was accomplished on the whole collection focusing on the gliadin fraction as main responsible for immunoreactivity in celiac disease (CD) patients. Complementary information about grain protein content, grain yield per spike, dry gluten and gluten index were collected. Cluster analysis was performed on original variables supporting the proper selection of five genotypes featuring medium and strong gluten strength, together with R5-reactivity and gliadin content lower than commercial semolina. Finally, the fate of gluten proteins was evaluated upon in-vitro simulated gastroduodenal digestion experiments carried out on raw wheat flours. The in-silico toxicity evaluation assessed a significantly lower number of toxic epitopes than commercial semolina.ConclusionsThe integrated approach confirmed that durum wheat breeding programs accomplished in the last decades improved the gluten strength, without causing an increment of toxic epitopes. Even if none of the selected genotypes can be considered safe for CD patients, a lower toxicity level could be envisaged and, in perspective, they may represent an innovative solution in genetically predisposed individuals who may develop CD after prolonged gluten consumption.

In this communication, we investigated the feasibility to develop an SPR based method tailored to the detection of egg residues in wines, the final goal being the elimination of matrix-effect on the analytical response. Two model wines matrices were selected, subjected to various purification procedures, and compared pair-wise with the standard curve both in terms of specific analytical responses and calibration curve slopes. Different statistical tools were used for significant comparisons.

In the present investigation, an LC-MS method for sensitive multiplex detection of five allergenic ingredients in a processed food matrix is presented. Cookie was chosen as complex food model and was incurred with egg, milk, soy, hazelnuts and peanuts before baking. Extraction, purification and pre-concentration protocols were applied to ground cookie basing on protocols described elsewhere. Specific instrumental features of a dual cell linear ion trap MS instrument were exploited to identify suitable peptide markers for each allergen and to deliver a sensitive multiplex SRM-based method for the simultaneous detection of common allergenic ingredients which might contaminate such a commodity.

There is a raising demand for sensitive and high throughput MS based methods for screening purposesespecially tailored to the detection of allergen contaminants in different food commodities. A challengingissue is represented by complex food matrices where the antibody-based kits commercially availablemight encounter objective limitations consequently to epitope masking phenomena due to a multitudeof interfering compounds arising from the matrix. The performance of a method duly optimized forthe extraction and simultaneous detection of soy, egg and milk allergens in a cookie food matrix by microHPLC-ESI-MS/MS, is herein reported. Thanks to the innovative configuration and the versatility shown by the dual cell linear ion trap MS used, the most intense and reliable peptide markers were first identified by untargeted survey experiment, and subsequently employed to design an ad hoc multitarget SRM method, based on the most intense transitions recorded for each selected precursor peptide. A sample extraction and purification protocol was optimized also including an additional step based on sonication, which resulted in a considerable improvement in the detection of milk allergen peptides. Data Dependent TM Acquisition scheme allowed to fill out a tentative list of potential peptide markers, which were further filtered upon fulfilling specific requirements. A total of eleven peptides were monitored simultaneously for confirmation purposes of each allergenic contaminant and the two most sensitive peptide markers/protein were selected in order to retrieve quantitative information. Relevant LODs were found to range from 0.1 g/g for milk to 0.3 g/g for egg and 2 g/g for soy.

A method based on High-Resolution Mass Spectrometry was developed for the simultaneousdetermination of fining agents containing potentially allergenic milk (casein) and egg-white (lysozyme and ovalbumin) proteins, added to commercial white wines at sub-ppm levels. Selected tryptic peptides were used as quantitative markers. An evaluation of protein digestion yields was also performed by implementing theanalogues of the best peptide markers identified for ? S1-casein and ovalbumin. The method was based on the combination of ultrafiltration (UF) of protein-containing wines, tryptic digestion of the dialyzed wine extracts and liquid chromatography/high resolution mass spectrometry (LC/HRMS) analysis of tryptic digests. Peptides providing the most intense electrospray ionization (ESI)-MS response were chosen as quantitative markers of the proteins under investigation. Six-point calibrations were performed by adding caseinate and egg-white powder in the concentration rangebetween 0.25 and 10 ug/mL, to an allergen-free white wine. The following three peptide markers, LTEWTSSNVMEER, GGLEPINFQTAADQAR and ELINSWVESQTNGIIR, were highlighted as best markers for ovalbumin, while GTDVQAWIR and NTDGSTDYGILQINSR for lysozyme and YLGYLEQLLR, GPFPIIV and FVAPFPEVFGK for caseinate. Limits of detection (LODs) ranged from 0.4 to 1.1 ug/mL. The developed method is suited for assessing the contemporary presence of allergenic milk and egg proteins characterizing egg white and caseinate, fining agents typically employed for wine clarification. The LODs of the method enable the detection of sub-ppm concentrations of residual fining agents, that could represent a potential risk for allergic consumers.

In questo contributo sono descritti i risultati della caratterizzazione spettroscopica di nanostrutture di oro ed argento, con diversa morfologia. I risultati ottenuti sulla composizione chimica delle superfici dei nanomateriali sono stati correlati alle loro prestazioni in spettrometria LDI-MS.

In the present, contribution angle-resolved X-ray photoelectron spectroscopy (AR-XPS) was proposed as a useful tool to address the challenge of probing the near-surface region of bio-active sensor surfaces. A model bio-functionalised surface was characterised by parallel AR-XPS and commercially available Thermo Avantage-ARProcess software was used to generate non-destructive concentration depth profiles of protein-functionalised silicon oxide substrates. At each step of the functionalisation procedure, the surface composition, the overlayer thickness, the in-depth organisation and the in-plane homogeneity were evaluated. The critical discussion of the generated profiles highlighted the relevance of the information provided by PAR-XPS technique. [Figure not available: see fulltext.] © 2012 Springer-Verlag.

In this paper we investigate a method for the detection of nanoparticles in order to reduce the risk associated with their toxicity, by taking into account the electromagnetic characteristics and the chemical analysis of the surface of a hybrid silicon photonic microresonator. Device sensing capabilities, both optical and chemical, are optimized in order to detect and size the nanoparticle. Thus, a silicon on insulator whispering gallery mode hybrid microresonator having an outer radius of 5 ?m and features that are typical of both ring and disk resonators, has been modeled. Quantum electrodynamics principles have been exploited in order to derive the master equation associated with the nanoparticle-resonator interaction. To allow a complete modeling of the sensor attention has been paid to the nanoparticle treatment, with the result that tested nanoparticles need to be chemically stabilized, monodisperse and formed by noble metal nanocolloids, in which a metal core (e.g. Au, Pd, etc) is surrounded by a monolayer or sub-monolayer film of an organic capping agent. © 2011 IEEE.

Food allergen research has made giant steps in the last years thanks to the features offered by the latest technology of mass analyzers placed on the market allowing multiplex sensitive detection of proteins. Potentials and features of two mass analyzers namely a linear ion trap capable of performing a data dependent or selected reaction monitoring analysis and an OrbitrapTM stand-alone MS enabling a broadband fragmentation without mass selection at highest mass resolving power are herein described and applied to the multiplex screening of allergens in a type of wine chosen as a reference matrix. Quantitative and confirmative capabilities of both platforms were assessed on the specific case study, the multiple detection of egg and milk-related proteins, typically employed in white wines as fining agents. Commercial bioinformatic tools used for a quick allergen identification will be also discussed.

The development of a surface plasmon resonance (SPR)-based biosensor tailored to the fast detection of egg-related fining allergens in wines is herein described. Ovalbumin (OVA) was chosen as the target protein to be monitored due to its highest abundance in the egg white powder, a typical fining agent used by the winery industry to promote wine clarification. A direct assay was designed, basing on the use of polyclonal anti-OVA antibody as bio-specific receptor. With the aim of optimizing the assay conditions, different parameters able to influence the final biosensor response were carefully investigated (i.e., pH, ionic strength, and additional surfactant concentration). After the fine tuning of these parameters, the assay was tested in the direct analysis of OVA in commercial wines artificially contaminated with egg white powder at different concentration levels in order to assess the reliability of the biosensor in detecting traces of OVA in complex matrices. The devised assay allowed to trace, in a short analysis time and with a minimal sample pre-treatment required, the presence of egg allergens at the lowest concentration comprised between 0.03 and 0.2 ?g/mL. Finally, the response provided by the developed biosensor was correlated with an established liquid chromatography mass spectrometry (LC-MS) method developed in our laboratories, and performances of both approaches were assessed for the fast monitoring of egg allergen contamination in fined wines.,[Figure not available: see fulltext.]

La resistenza delle proteine allergeniche della soia in seguito a digestione gastro-duodenale simulata sarà discussa nella presente comunicazione unitamente all'uso di software di bioinformatica per valutare l'allergenicità residua.

Over the last years, great efforts have been devoted to develop effective gluten detoxification strategies with a consequent detrimental alteration of the technological properties as well. Obtaining low-gluten products without affecting the rheological properties of wheat could still be considered a new challenge to face.In this investigation, we presented a comprehensive characterization of durum wheat genotypes aimed at identifying low gluten ones, which combine the potential lower toxicity/immunogenicity with conserved yield and rheological properties to encompass the perspective usability for bread or pasta making. A preliminary profiling of gluten proteins was accomplished by immunoassay-based quantification and liquid chromatography coupled to UV detection, focusing on the gliadin fraction as main responsible for immunoreactivity in celiac disease patients. In addition, data on grain protein content, grain yield per spike, dry gluten and gluten index were collected in order to provide complementary information about productivity-related traits and quali-quantitative characteristics related to wheat nutritional value and its technological properties. The whole pool of data was statistically evaluated driving to the selection of a preferred list of candidate low-toxicity genotypes that were subjected to in-vitro simulated gastroduodenal digestion and untargeted HR-MS/MS peptide identification. Finally, an in-silico risk assessment of potential toxicity for celiac disease patients was performed according to the most recent guidance provided by EFSA.

Contamination of food products by allergens represents a matter of concern especially for allergic consumers due to the risk of triggering an immunological reaction upon ingestion of allergen-containing foods [1]. Due to the widespread extent of such pathology, and in order to protect the health of sensitive consumers, specific legislation has been issued in different countries on the proper labeling of a restricted list of food allergens whenever added to food [2]. Besides their intentional incorporation into the commodity, a risk of accidental contamination is likely to exist. In this case allergens are defined hidden as they have not been declared on the product label and might unexpectedly reach the end products through several routes [3]. Different analytical methods have been developed in the last years for monitoring food allergen contamination along the food chain. Recently, mass spectrometry (MS) methods [1], have been considered a promising analytical strategy for f ood allergens detection thanks to the advances made in this technology that enables to overcome several restrictions of antibody-based methods, such as ELISA. Among them the risk of false positives, especially when applied to complex or processed food matrices that might cause epitope modification or masking, and the limitations in multiplexing. In the present investigation, a sensitive LC-MS method tailored to the multiplex detection of several allergenic ingredients in a processed food matrix will be described. Cookie was chosen as complex and processed food model, incurred with egg, milk, soy, hazelnut and peanut allergens. Starting from our previous investigation [4], extraction, purification and enzymatic digestion conditions were duly optimized in order to design a relatively fast and easy sample handling procedure which allowed to considerably reduce the time requested for sample preparation before LC-MS analysis. Features of two different MS instrumental set-up, also exploiting High Resolution Mass Spectrometric detection, to monitor 5 allergenic foods in complex foods will be presented. An automated on line pre-enrichment procedure onto a trap-column followed by chromatographic separation was designed enabling the pre-enrichment and partial purification of the candidate markers with challenging LODs obtained.

Allergenic ingredients in pre-packaged foods are regulated by EU legislation mandating their inclusion on labels. In order to protect allergic consumers, sensitive analytical methods are required for detect allergen traces in different food products. As a follow-up to our previous investigations, an optimized, sensitive ,label-free LC-MS/MS method for multiplex detection of five allergenic ingredients in a processed food matrix is proposed. A cookie base was chosen as a complex food matrix and home-made cookies incurred with whole egg, skimmed milk, soy flour, ground hazelnut and ground peanut were prepared at laboratory scale. In order to improve the analytical workflow both protein extraction and purification protocols were optimized in order to achieve a sensitive streamlined SRM based analytical method for allergens detection in incurred cookies. The effect of baking on the detection of selected markers was also investigated .

Metal nanomaterials have an emerging role in surface-assisted laser desorption ionisation-mass spectrometry (SALDI-MS) providing a useful tool to overcome some limitations intrinsically related to the use of conventional organic matrices in matrix-assisted LDI-MS. In this contribution, the possibility to use a stainless-steel-supported gold nanoparticle (AuNP) film as a versatile platform for SALDIMS was assessed. A sacrificial anode electrosynthetic route was chosen in order to obtain morphologically controlled core-shell AuNPs; the colloidal AuNPs were, thereafter, drop cast onto a stainless-steel sample plate and the resulting AuNP film was thermally annealed in order to improve its effectiveness as LDI-MS promoter. Spectroscopic characterization of the nanostructured film by X-ray photoelectron spectroscopy was crucial for understanding how annealing induced changes in the surface chemistry and influenced the performance of AuNPs as desorption/ionisation promoter. In particular, it was demonstrated that the post-deposition treatments were essential to enhance the AuNP core/analyte interaction, thus resulting in SALDI-MS spectra of significantly improved quality. The AuNP films were applied to the detection of three different classes of low molecular weight (LMW) analytes, i.e. amino acids, peptides and LMW polymers, in order to demonstrate the versatility of this nanostructured material. © Springer-Verlag 2012.

Untargeted HRMS approach for food authenticity: Discrimination of salmon (Salmo Salar) living conditions and geographical origin

Saffron is a valuable and highly appreciated spice derived from the dried red stigmas of the flowers of the cultivated plant Crocus sativus L. It is commonly used as a coloring and flavouring agent in food preparation. In addition, it is a good source of flavonoids, proteins, sugars, vitamins, amino acids, mineral matter, gums, and other chemical compounds that make it a health promoting spice. To produce saffron spice, harvested stigmas are submitted to a mild food processing peculiar of the region of production thus it could be considered a traditional product, which aroma and chemical composition strictly depend on the geographic location of production. Due to its high costs of production, saffron is one of the most expensive spice commercialized across the world, and often susceptible of adulteration. Different plant-derived adulterants have been discovered, and the most frequently involve cut and/or dyed Carthamus tinctorius L. petals and Curcuma longa L. powdered rhizomes (Kanti et al., 2011). Several analytical methods have been reported in literature for the detection of plant adulterants in saffron samples based on different techniques (e.g. Maggi et al., 2011, Zalacain et al., 2005, Tarantilis et al., 2004, Zougagh et al., 2005) although official methods (ISO 3632-1; ISO 3632-2) exist. Very recently, high-performance liquid chromatography coupled to high resolution mass spectrometry (HRMS) was successfully proposed for saffron authentication/traceability according to the geographical origin based on untargeted metabolic fingerprinting (Rubert et al., 2016). In this work, we investigated two HRMS based approaches one using ESI ionization coupled to LC separation and the other based on DART ionization before HRMS detection to assess saffron authenticity by an untargeted metabolic approach. Pure saffron samples and saffron spiked with different amounts of Carthamus and Curcuma adulterants were extracted with a mix of ethanol and water to extract the majority of metabolites and final MS fingerprints obtained were used for authenticity assessment and/or adulteration detection. The produced spectra were then processed via the commercial software Compound Discoverer v.2.1 SP1 (Thermo Fisher Scientific). The detection and grouping of the unknown compounds by setting a mass accuracy <= 5ppm was further accomplished, together with a preliminary statistical analysis of the integrated peak areas. As grouping factor "Type of adulterant" (Carthamus and Curcuma) was set and data were further pretreated by aligning the extracted chromatograms on the respective retention time. In order to filter the compound list to the species most suitable in discriminating pure saffron from the adulterated one, Volcano plots combining the statistical significance of the identified compounds and magnitude of change in the extracted peak areas, were investigated. Edited compounds list constrained by means of p-value thresholds was then subjected to statistical evalua

In the present contribution, the potential of LC-HR-MS coupled to software based data handling for fish authenticity and traceability will be presented. Salmon salar discrimination based on geographic origin was accomplished by untargeted LC-HR-MS analysis on hybrid quadrupole/OrbitrapTM-based mass spectrometer and by raw data mining with commercial software for small molecules identification. Salmon filets were extracted by methanol/chloroform mixtures and both the polar and non-polar fractions were analysed for a comprehensive compound characterization and differential analysis. In particular, conventional chromatographic separation was accomplished with a relatively short linear gradient with full scan HR-MS acquisitions performed in both positive and negative modes. The gathered spectra were processed via the commercial software Compound Discoverer v.2.0 (Thermo Fisher Scientific) in order to detect and group the unknown compounds with an accuracy <= 2ppm and achieve a preliminary statistical analysis of the integrated peak areas. The study was carried out by setting as grouping factor the geographic origin (Canada, Norway and Chile) and data were pretreated by means of the retention time alignment of extracted chromatograms and background subtraction. Volcano plots combining the statistical significance of the identified compounds and magnitude of change in the extracted peak areas were investigated in order to filter the compound list to the main species most suitable in discriminating the different salmon groups. Edited compounds list constrained by means of p-value thresholds was subjected within the same workflow to further statistical evaluation by PCA in order to simplify the multivariate system and highlight the intrinsic trend of the analyzed samples in the reduced projected space. The PCA performed on polar fraction of the salmon extracts allowed to distinguish clearly three data groups belonging to the three geographic origins (Canada, Norway and Chile).

Composite films composed of poly(3,4-ethylenedioxythiophene), PEDOT, and the filamentous virus M13-K07 were prepared by electrooxidation of 3,4-ethylenedioxythiophene (EDOT) in aqueous solutions containing 8 nM of the virus at planar gold electrodes. These films were characterized using atomic force microscopy and scanning electron microscopy. The electrochemical impedance of virus-PEDOT films increases upon exposure to an antibody (p-Ab) that selectively binds to the M13 coat peptide. Exposure to p-Ab causes a shift in both real (Z RE) and imaginary (Z IM) impedance components across a broad range of frequencies from 50 Hz to 10 kHz. Within a narrower frequency range from 250 Hz to 5 kHz, the increase of the total impedance (Z total) with p-Ab concentration conforms to a Langmuir adsorption isotherm over the concentration range from from 6 to 66 nM, yielding a value for K d = 16.9 nM at 1000 Hz.