Development of a Mass Spectrometry-Based Method for Quantification of Total Cashew Protein in Roasting Oil.

BACKGROUND: Food allergen cross-contact during food preparation and production is one of the causes of unintentional allergen presence in packaged foods. However, little is known about allergen cross-contact in shared frying or roasting oil, which prevents the establishment of effective allergen controls and may put allergic individuals at risk. To better understand the quantity of allergen transferred to frying oil and subsequent products, an analytical method is needed for quantifying protein in oil that has been exposed to frying/roasting conditions. OBJECTIVE: The goal of this study was to develop a parallel reaction monitoring LC-MS/MS method to quantify the amount of cashew protein in shared roasting oil. METHODS: The sample preparation method was evaluated to improve protein extractability and peptide performance. Four quantitative peptides representing cashew 2S and 11S proteins were selected as targets based on their sensitivity, heat stability, and specificity. A calibration strategy was developed to quantify the amount of total cashew protein in oil. Method performance was evaluated using a heated cashew-in-oil model system. RESULTS: The method showed high recovery in oil samples spiked with 100 or 10 parts per million (ppm) total cashew protein heated at 138 or 166°C for 2-30 min. Samples (100 ppm total cashew protein) heated for 30 min had more than 90% recovery when treated at 138°C and more than 50% when heated at 166°C. CONCLUSION: The method is fit-for-purpose for the analysis of cashew allergen cross-contact in oil. HIGHLIGHTS: A novel MS-based method was developed that can accurately quantify the amount of cashew protein present in heated oil.

Proteomic Analysis of Oil-Roasted Cashews Using a Customized Allergen-Focused Protein Database.

Cashews are one of the most prevalent causes of tree nut allergies. However, the cashew proteome is far from complete, which limits the quality of peptide identification in mass spectrometric analyses. In this study, bioinformatics tools were utilized to construct a customized cashew protein database and improve sequence quality for proteins of interest, based on a publicly available cashew genome database. As a result, two additional isoforms for cashew 2S albumins and five other isoforms for cashew 11S proteins were identified, along with several other potential allergens. Using the optimized protein database, the protein profiles of cashew nuts subjected to different oil-roasting conditions (139 and 166 °C for 2-10 min) were analyzed using discovery LC-MS/MS analysis. The results showed that the cashew 2S protein is most heat-stable, followed by 11S and 7S proteins, though protein isoforms might be affected differently. Preliminary target peptide selection indicated that out of the 29 potential targets, 18 peptides were derived from the newly developed database. In the evaluation of thermal processing effects on cashew proteins, several Maillard reaction adducts were also identified. The cashew protein database developed in this study allows for comprehensive analyses of cashew proteins and development of high-quality allergen detection methods.

Development and Interlaboratory Evaluation of an LC-MS/MS Method for the Quantification of Lysozyme in Wine Across Independent Instrument Platforms.

BACKGROUND: Various processing aids and fining agents are used in winemaking to help improve sensory characteristics. Some of these materials may contain or be derived from allergenic foods, such as eggs. In order to ensure food safety and that products meet regulatory compliance, it is essential to have robust and effective analytical methods to verify the removal of allergenic proteins following their use. Current methods include ELISA and MS methods, which can target either whole foods or individual proteins, and provide either quantitative data or qualitative confirmation of proteins. MS methods offer the potential to test for multiple proteins within a single assay to improve cost and efficiency, whereas ELISA methods typically analyze for a single protein per assay. OBJECTIVE: This study focuses on the development of a LC-tandem MS (MS/MS) quantitative method for lysozyme in white wine and compares performance across two laboratories utilizing two different instrument platforms. METHODS: Lysozyme target peptides were selected by conducting bottom-up discovery proteomics. Candidate targets were evaluated using parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) LC-MS/MS, depending on the instrument in each laboratory. Quantification of lysozyme was conducted using internal, stable isotope-labeled synthetic peptide standards. RESULTS: Three of eight candidate target peptides showed performance suitable for the final quantitative method. White wine spiked with 0.1 and 0.5 ppm lysozyme demonstrated quantitative recovery of 70-120%. While the PRM method delivered better repeatability, the SRM method gave higher quantitative recovery values. CONCLUSION: A targeted LC-MS/MS method for quantification of lysozyme in white wine has been developed and deployed on two different MS instrument platforms in two laboratories. HIGHLIGHTS: Both SRM and PRM targeted LC-MS/MS methodologies can be used for quantification of lysozyme in white wine. This study is among the first to evaluate an MS method for food allergen quantification in multiple laboratories.

Quantification of Soy-Derived Ingredients in Model Bread and Frankfurter Matrices with an Optimized Liquid Chromatography-Tandem Mass Spectrometry External Standard Calibration Workflow.

ABSTRACT: The detection and quantification of soy protein is important for food allergen management and identifying the presence of undeclared soy proteins. Heat processing and matrix interactions can affect the accuracy of allergen detection methods. The sensitivity of enzyme-linked immunosorbent assay methods can be compromised if protein epitopes are modified during processing. Therefore, a mass spectrometry (MS)-based method was evaluated for the recovery of total soy protein in incurred matrices. MS-based quantification of total soy protein was assessed by using a combination of external and internal standards. The reproducibility of the standard curves was investigated by comparing within-day and among-day variation. Incurred samples were prepared using bread and frankfurters as model food matrices. Several soy-derived ingredients were used to prepare the matrices with varying levels of soy protein (1, 10, 50, or 100 ppm of total soy protein). A pooled standard curve was used to estimate the total soy protein concentration of the incurred food matrices and the percent total protein recovery. The variation of replicate standard curves between days and among all days was not significant. The differences in slopes obtained from replicate standards run on different days were minimal. The most influential factor on the quantitative protein recovery in incurred samples was the effect of the physical matrix structure on protein extraction. The lowest percent protein recoveries, less than 50%, were calculated for uncooked matrices. The cooked matrices had percentage recoveries between 50 and 150% for all total soy protein levels. Other factors, such as type of ingredient, were determined to be not as impactful on recovery. The MS method described in this study was able to provide sensitive detection and accurate quantification of total soy protein from various soy-derived ingredients present in processed food matrices.

Gluten Cross-Contact in Restaurant-Scale Pasta Cooking.

ABSTRACT: Celiac disease and nonceliac gluten sensitivity are provoked by the consumption of gluten from wheat, barley, rye, and related grains. Affected individuals are advised to adhere to gluten-free diets. Recently, gluten-free foods have become a marketing trend with gluten-free options both in packaged foods and in restaurants and food service establishments. Pasta is one of the primary gluten-containing foods in diets in North America and Europe. Gluten-free pasta formulations are commercially available. In restaurants, multiple pasta dishes are often prepared simultaneously in large pots with multiple compartments and shared cooking water. The objective of this study was to determine whether gluten transfer occurs between traditional and gluten-free pasta when cooked simultaneously in the same water. Pasta was boiled in a commercial, four-compartment, 20-qt (18.9-L) cooking pot containing three batches of traditional penne pasta and one batch of gluten-free penne pasta. The amount of pasta (dry weight) was either 52 g (recommended serving size) or 140 g (typical restaurant portion). Five consecutive batches of pasta were boiled, and cooking water and gluten-free pasta were sampled at completion of cooking. Water and gluten-free pasta samples were tested for gluten with the Neogen Veratox for Gliadin enzyme-linked immunosorbent assay kit. Gluten concentrations were low (<20 ppm) in both water and gluten-free pasta samples through five 52-g batches. Gluten concentrations in the 52-g gluten-free pasta samples slowly increased through five batches but were never >20 ppm. During cooking of the 140-g gluten-free pasta samples, the gluten concentrations in the cooking water increased with each batch to >50 and >80 ppm after the fourth and fifth batches, respectively. The gluten concentrations in the 140-g gluten-free pasta samples approached 20 ppm by the fourth batch and reached nearly 40 ppm after the fifth batch. Although gluten transfer does not occur at a high rate, gluten-free pasta should be prepared in a separate cooking vessel in restaurant and food service establishments.

Tracking Gluten throughout Brewing Using N-Terminal Labeling Mass Spectrometry.

Gluten-containing grains cause adverse health effects in individuals with celiac disease. Fermentation of these grains results in gluten-derived polypeptides with largely uncharacterized sizes and sequences, which may still trigger an immune response. This research used N-terminal labeling mass spectrometry to characterize protein hydrolysates during each stage of bench-scale brewing, including malting, mashing, boiling, fermentation, and aging. Gluten hydrolysates from each brewing step were tracked, and the immunotoxic potential was evaluated by sequence comparison with peptides known to stimulate celiac immune responses. The results indicate that proteolysis and precipitation of gliadins occurring during brewing differ by protein region and brewing stage. The termini of gliadins were hydrolyzed throughout the entire brewing process, but the central regions remained relatively stable. Most hydrolysis occurred during malting, and most precipitation occurred during boiling. The addition of yeast yielded new cleavage sites but did not result in complete hydrolysis. Consistent detection of peptides within the clinically important regions of gliadin corroborated the hydrolytic resistance of this region. N-terminal labeling mass spectrometry served as a novel approach to track the fate of gliadin/gluten throughout bench-scale brewing. Consistently identified fragments could serve as improved targets for the detection of hydrolyzed gluten in fermented products.

Compositional and immunogenic evaluation of fractionated wheat beers using mass spectrometry.

The safety and regulatory status of fermented products derived from gluten-containing grains for patients with celiac disease remains controversial. Bottom-up mass spectrometry (MS) has complemented immunoassays for the compositional and immunogenic analyses of wheat beers. However, uncharacterized proteolysis during brewing followed by the secondary digestion for MS has made the analysis and data interpretation complicated. In this study, the composition and immunogenic potential of seven commercially available wheat beers were evaluated using bottom-up MS with the aid of fractionation and a multi-step peptide search strategy to identify peptides generated by various types of proteolysis. Gluten-derived peptides accounted for approximately 50% and 20% of the total number of wheat-derived and barley-derived peptides, respectively, in the investigated beers. Although relatively large polypeptides cannot be thoroughly characterized using traditional bottom-up proteomics, up to 50% of peptides identified contained celiac-immunogenic motifs, and consumption of wheat beers would pose risks for celiac patients.

Parallel Reaction Monitoring Mass Spectrometry Method for Detection of Both Casein and Whey Milk Allergens from a Baked Food Matrix.

Milk allergy is among the most common food allergies present in early childhood, which in some cases may persist into adulthood as well. Proteins belonging to both casein and whey fractions of milk can trigger an allergic response in susceptible individuals. Milk is present as an ingredient in many foods, and it can also be present as casein- or whey-enriched milk-derived ingredients. As whey proteins are more susceptible to thermal processing than caseins, conventional methods often posed a challenge in accurate detection of whey allergens, particularly from a processed complex food matrix. In this study, a targeted mass spectrometry method has been developed to detect the presence of both casein and whey allergens from thermally processed foods. A pool of 19 candidate peptides representing four casein proteins and two whey proteins was identified using a discovery-driven target selection approach from various milk-derived ingredients. These target peptides were evaluated by parallel reaction monitoring of baked cookie samples containing known amounts of nonfat dry milk (NFDM). The presence of milk could be detected from baked cookies incurred with NFDM at levels as low as 1 ppm using seven peptides representing α-, ß-, and κ-casein proteins and three peptides representing a whey protein, ß-lactoglobulin, by this consensus PRM method.

Evaluation of N-terminal labeling mass spectrometry for characterization of partially hydrolyzed gluten proteins.

Gluten, a group of proteins found in wheat, barley, and rye, is the trigger of celiac disease, an immune disorder that affects about 1% of people worldwide. The toxicity of partially hydrolyzed gluten (PHG) in fermented products is less well understood due to the significant analytical challenges in PHG characterization. In this project, an N-terminal labeling mass spectrometry method, terminal amine isotopic labeling of substrates (TAILS), was optimized for the in-depth analysis of PHG and validated using a test protease (trypsin) with known cleavage specificity. Gluten N-termini in test and control groups were labeled with heavy and light formaldehyde, respectively. Trypsin-generated neo N-termini were identified by exhibiting an MS1 Log2 H:L peak area ratio with a significant difference (p < .01) from zero and native N-termini with no significant difference from zero (p > .01). Using this strategy, all abundant, theoretical, test protease-generated peptides in exemplar alpha/beta gliadins and gamma gliadins were identified. SIGNIFICANCE: This study is the first study that modified and evaluated TAILS analysis for the analysis of partially hydrolyzed gluten proteins. The evaluation indicated that the TAILS analysis could be modified and expanded to the identification of multiple protease cleavage sites in gluten proteins and is worth further evaluation as a novel strategy for the analysis of natural hydrolysis of gluten in food processes. This strategy also may be further applied to characterize a broader range of partially hydrolyzed allergens in foods and provide reference for their safety assessment to both industry and regulatory authorities.

Detection of Six Commercially Processed Soy Ingredients in an Incurred Food Matrix Using Parallel Reaction Monitoring.

Soybeans are one of the major allergenic foods in many countries. Soybeans are commonly processed into different types of soy ingredients to achieve the desired properties. The processing, however, may affect the protein profiles and protein structure, thus affecting the detection of soy proteins. Mass spectrometry (MS) is a potential alternative to the traditional immunoassays for the detection of soy-derived ingredients in foods. This study aims to develop a liquid chromatography-tandem MS method that uniformly detects different types of soy-derived ingredients. Target peptides applicable to the detection of six commercial soy ingredients were identified based on the results of MS label-free quantification and a set of selection criteria. The results indicated that soy ingredient processing can result in different protein profiles. A total of six soy ingredients were then individually incurred into cookie matrices at different levels. Sample preparation methods were optimized, and a distinct improvement in peptide performance was observed after optimization. Cookies and dough incurred with different soy ingredients at 100 ppm total soy protein showed a similar level of peptide recovery (90% mean signal relative to unroasted soy flour), demonstrating the ability of the MS method to detect processed soy ingredients in a uniform manner.

Target Selection Strategies for LC-MS/MS Food Allergen Methods.

The detection and quantitation of allergens as contaminants in foods using MS is challenging largely due to the requirement to detect proteins in complex, mixed, and often processed matrixes. Such methods necessarily rely on the use of proteotypic peptides as indicators of the presence and amount of allergenic foods. These peptides should represent the allergenic food in question in such a way that their use is both sensitive (no false-negatives) and specific (no false-positives). Choosing such peptides to represent food allergens is beset with issues, including, but not limited to, separated ingredients (e.g., casein and whey), extraction difficulties (particularly from thermally processed foods), and incomplete sequence information, as well as the more common issues associated with protein quantitation in biological samples. Here, we review the workflows that have been used to select peptide targets for food allergen detection. We describe the use and limitations of both in silico-based analyses and experimental methods relying on high-resolution MS. The variation in the way in which target selection is performed highlights a lack of standardization, even around the principles describing what the detection method should achieve. A lack of focus on the food matrixes to which the method will be applied is also apparent during the peptide target selection process. It is hoped that highlighting some of these issues will assist in the generation of MS-based allergen detection methods that will encourage uptake and use by the analytical community at large.

Mass spectrometric analysis of allergens in roasted walnuts.

UNLABELLED: Thermal processing of allergenic foods can induce changes in the foods' constituent allergens, but the effects of heat treatment are poorly defined. Like other commonly allergenic tree nuts, walnuts often undergo heat treatment (e.g. roasting or baking) prior to consumption. This study evaluated the changes in solubility and detectability of allergens from roasted walnuts using tandem mass spectrometry methods. Walnuts were roasted (132°C or 180°C for 5, 10, or 20min) and prepared for LC-MS/MS using sequential or simultaneous extraction and tryptic digestion protocols. The LC-MS/MS data analysis incorporated label-free quantification of relevant allergens and Maillard adduct screening. In some proteins (2S albumin, LTP, and the 7S globulin N-terminal region) minor changes in relative abundance were observed following roasting. The mature 7S and 11S globulins, however, showed significantly increased detection following roasting at 180°C for 20min when using the simultaneous extraction/digestion protocol, possibly due to increased digestibility of the proteins. The results of this study indicate that individual walnut allergens respond differently to thermal processing, and the detection of these proteins by LC-MS/MS is dependent on the protein in question, its susceptibility to proteolytic digestion, the degree of thermal processing, and the sample preparation methodology. SIGNIFICANCE: Understanding the behavior of food allergens in the context of relevant food matrices is critical for both food allergen management and for elucidating matrix and processing-associated factors influencing protein allergenicity. The use of mass spectrometry to identify food allergens and detect allergenic food residues has been increasingly developed due to the advantages associated with the direct, sequence-level analysis possible with MS. To date, however, few studies have implemented MS technology to analyze the effects of thermal processing on allergenic food proteins. The MS analysis results presented in this study revealed not only information about the molecular-level effects of roasting on walnut allergens but also data pertinent to the development of MS-based detection methods for walnut residues in food products.

Insoluble and soluble roasted walnut proteins retain antibody reactivity.

Thermal processing techniques commonly used during food production have the potential to impact food allergens by inducing physical and/or chemical changes to the proteins. English walnuts (Juglans regia) are among the most commonly allergenic tree nuts, but little information is available regarding how walnut allergens respond to thermal processing. This study evaluated the effects of dry roasting (132 or 180°C for 5, 10, or 20min) on the solubility and immunoreactivity of walnut proteins. A dramatic decrease in walnut protein solubility was observed following dry roasting at 180°C for 20min. However, both the soluble and insoluble protein fractions from roasted walnuts maintained substantial amounts of IgG immunoreactivity (using anti-raw and anti-roasted walnut antisera), with similar patterns of reactivity observed for human IgE from walnut-allergic individuals. Thus, walnut proteins are relatively stable under certain thermal processing conditions, and IgE reactivity remains present even when insoluble aggregates are formed.

Characterization of low molecular weight allergens from English walnut (Juglans regia).

Although English walnut is a commonly allergenic tree nut, walnut allergens have been poorly characterized to date. The objective of this work was to characterize the natural, low molecular weight (LMW) allergens from walnut. A protocol was developed to purify LMW allergens (specifically 2S albumins) from English walnuts. In addition to 2S albumins, a series of peptides from the N-terminal region of the 7S seed storage globulin proprotein were also identified and characterized. These peptides comprised a four-cysteine motif (C-X-X-X-C-X10-12-C-X-X-X-C) repeated throughout the 7S N-terminal region. Upon IgE immunoblotting, 3/11 and 5/11 sera from walnut-allergic subjects showed IgE reactivity to the 7S N-terminal fragments and 2S albumin, respectively. The mature 7S protein and the newly described 7S N-terminal peptides represent two distinct types of allergens. Because the proteolytic processing of 7S globulins has not been elucidated in many edible plant species, similar protein fragments may be present in other nuts and seeds.

Effects of thermal processing on the enzyme-linked immunosorbent assay (ELISA) detection of milk residues in a model food matrix.

Food products and ingredients are frequently tested for the presence of undeclared allergenic food residues (including milk) using commercial enzyme-linked immunosorbent assays (ELISAs). However, little is understood about the efficacy of these kits with thermally processed foods. This study evaluated the performance of three milk ELISA kits with a model food processed by several methods. A model food (pastry dough squares) was spiked with nonfat dry milk at several concentrations. The pastry squares were processed by boiling (100 °C for 2 min), baking (190 °C for 30 min), frying (190 °C for 2 min), and retorting (121 °C for 20 min with 17 psi overpressure). Samples were analyzed with three commercial ELISA kits: Neogen Veratox Total Milk, ELISA Systems ß-lactoglobulin, and ELISA Systems casein. The detection of milk residues depended upon the type of processing applied to the food and the specific milk analyte targeted by the ELISA kit. Poor recoveries were obtained in all processed samples (2-10% of expected values) using the ß-lactoglobulin kit. Better recoveries were obtained in boiled samples (44 and 59%, respectively) using the total milk and casein kits. However, these kits performed poorly with baked (9 and 21%) and fried (7 and 18%) samples. Moderate recoveries were observed in retorted samples (23 and 28%). The decreased detection in processed samples is likely due to protein modifications, including aggregation and Maillard reactions, which affect the solubility and immunoreactivity of the antigens detected by the ELISA methods. The observed decreases in ELISA detection of milk are dramatic enough to affect risk-assessment decisions. However, a lower detection of milk residues does not necessarily indicate decreased allergenicity. These ELISA kits are not acceptable for all applications, and users should understand the strengths and limitations of each method.