Rosa Pilolli

Multi-allergen detection in food by micro high-performance liquid chromatography coupled to a dual cell linear ion trap mass spectrometry

There is a raising demand for sensitive and high throughput MS based methods for screening purposes especially tailored to the detection of allergen contaminants in different food commodities. A challenging issue is represented by complex food matrices where the antibody-based kits commercially available might encounter objective limitations consequently to epitope masking phenomena due to a multitude of interfering compounds arising from the matrix. The performance of a method duly optimized for the 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 multi-target 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™ 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 0.1μg/g for milk to 0.3μg/g for egg and 2μg/g for soy.

Multi‐allergen quantification of fining‐related egg and milk proteins in white wines by high‐resolution mass spectrometry

RATIONALE A method based on High-Resolution Mass Spectrometry was developed for the simultaneous determination 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 the (15)N-valine-labelled analogues of the best peptide markers identified for αS1 -casein and ovalbumin. METHODS 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. RESULTS Six-point calibrations were performed by adding caseinate and egg-white powder in the concentration range between 0.25 and 10 µg/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 FFVAPFPEVFGK for caseinate. Limits of detection (LODs) ranged from 0.4 to 1.1 µg/mL. CONCLUSIONS 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.

Orbitrap™ monostage MS versus hybrid linear ion trap MS: application to multi-allergen screening in wine.

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 Orbitrap(TM) 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.

Thermally annealed gold nanoparticles for surface-assisted laser desorption ionisation-mass spectrometry of low molecular weight analytes

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 SALDI-MS 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.

High-resolution Orbitrap™-based mass spectrometry for rapid detection of peanuts in nuts.

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™ 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™ 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. Graphical Abstract

Streamlining the analytical workflow for multiplex MS/MS allergen detection in processed foods

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 and finally a sensitive streamlined SRM based analytical method for allergens detection in incurred cookies was devised. The effect of baking on the detection of selected markers was also investigated.

Investigation of Heat and Pressure Treatments on Almond Protein Stability and Immunoreactivity after Simulated Human Digestion

Almond is worldwide consumed and renowned as a valuable healthy food. In spite of this, 14 it is also a potent source of allergenic proteins able to trigger several mild to life-threatening 15 immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus 16 affecting the respective allergenicity. In this paper we investigated the effect of autoclaving, preceded 17 or not by a hydration step, on the biochemical and immunological properties of almond proteins. 18 Any variation in the stability and immunoreactivity of almond proteins extracted from the treated 19 materials, were evaluated by total protein quantification, ELISA assay and protein profiling by 20 electrophoresis-based separation (SDS-PAGE). The autoclaving alone was found to weakly affect 21 almond proteins stability, despite what observed for the combination of hydration and autoclaving, 22 which resulted in a loss of approximately 70% of total protein content compared to untreated sample, 23 and in a final negligible immunoreactivity, as well. The final SDS-PAGE protein pattern recorded for 24 almonds hydrated and autoclaved disclosed significant changes. In addition, the same samples were 25 further submitted to in vitro simulated gastro-duodenal (GI) digestion to evaluate potential changes 26 induced by these processing on allergens digestibility. Digestion products were identified by HPLC27 HRMS/MS analysis followed by software-based data mining, and complementary information were 28 provided by analyzing the proteolytic fragments lower that 6 kDa in size. The autoclave based 29 treatment was found not to alter the allergens digestibility, whereas an increased susceptibility to 30 proteolytic action of digestive enzymes was observed in almonds subjected to the combination of 31 prehydration and autoclaving. Finally, the residual immunoreactivity of the GI resistant peptides 32 was investigated in-silico by bioinformatic tools, confirming that by following both approaches, no 33 epitopes survived the almond digestion, thus demonstrating the potential effectiveness of these 34 treatments to reduce almond allergenicity. 35

Effects of the Varietal Diversity and the Thermal Treatment on the Protein Profile of Peanuts and Hazelnuts

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.