Identification of Cross-Species Marker Peptides for the Detection of Mammalian and Poultry Meat in Vegan and Vegetarian Foods.

Authentication of vegan and vegetarian foods is important since these increasingly popular food items could be adulterated with cheap meat to increase profit margins. In this study, nine marker peptides for the detection of meat (several species) were identified applying liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). These marker peptides enable the crucial differentiation of beef versus milk and chicken meat versus egg, demonstrated by the investigation of 19 commercial vegetarian meat substitutes containing milk and egg. Extensive experimental testing proved the presence of the cross-species meat marker peptides in 19 food-relevant types of mammals and poultry as well as their absence in more than 136 plant-based ingredients for the production of vegan and vegetarian foods. An authentic vegan sausage matrix based on an actual retail product was produced and spiked with 5.0%, w/w meat to confirm the high signal intensities and the heat stability of the marker peptides.

Genome-wide deposition of 6-methyladenine in human DNA reduces the viability of HEK293 cells and directly influences gene expression.

While cytosine-C5 methylation of DNA is an essential regulatory system in higher eukaryotes, the presence and relevance of 6-methyladenine (m6dA) in human cells is controversial. To study the role of m6dA in human DNA, we introduced it in human cells at a genome-wide scale at GANTC and GATC sites by expression of bacterial DNA methyltransferases and observed concomitant reductions in cell viability, in particular after global GANTC methylation. We identified several genes that are directly regulated by m6dA in a GANTC context. Upregulated genes showed m6dA-dependent reduction of H3K27me3 suggesting that the PRC2 complex is inhibited by m6dA. Genes downregulated by m6dA showed enrichment of JUN family transcription factor binding sites. JUN binds m6dA containing DNA with reduced affinity suggesting that m6dA can reduce the recruitment of JUN transcription factors to target genes. Our study documents that global introduction of m6dA in human DNA has physiological effects. Furthermore, we identified a set of target genes which are directly regulated by m6dA in human cells, and we defined two molecular pathways with opposing effects by which artificially introduced m6dA in GANTC motifs can directly control gene expression and phenotypes of human cells.

Characterization and Quantification of Mal d 1 Isoallergen Profiles and Contents in Traditional and Commercial Apple Varieties by Mass Spectrometry.

The apple allergy in Northern Europe is a cross-reaction to the birch pollen allergy. No correlation between the allergenicity of an apple variety and the content of the major apple allergen Mal d 1, a homologue to the Bet v 1 allergen in birch, could be found using ELISA, so far. Therefore, an impact of polyphenols and/or differences in the isoallergen profile are discussed. To allow a more detailed analysis of the Mal d 1 content and the isoallergen profile, a mass spectrometric method was applied to investigate differences in the flesh and peel of 10 traditional varieties and 10 commercial breeds. The data revealed often, but not always, lower Mal d 1 contents in traditional varieties grown in orchard meadows, which was more obvious in the flesh. Differences among the peels were less pronounced. A closer look at the individual isoallergens 1.01, 1.02, 1.03, and 1.06 reveals an increased impact of the minor isoallergens 1.03 and 1.06 on the allergenic potential, since commercial breeds like Braeburn, Santana, and Holstein Cox, which are considered to have reduced allergenic potentials, were characterized by low levels of these isoallergens.

The R736H cancer mutation in DNMT3A modulates the properties of the FF-subunit interface.

The DNMT3A DNA methyltransferase is an important epigenetic enzyme that is frequently mutated in cancers, particularly in AML. The heterozygous R736H mutation in the FF-interface of the tetrameric enzyme is the second most frequently observed DNMT3A cancer mutation, but its pathogenic mechanism is unclear. We show here that R736H leads to a moderate reduction in catalytic activity of 20-40% depending on the substrate, but no changes in CpG specificity, flanking sequence preferences and subnuclear localization. Strikingly, R736H showed a very strong stimulation by DNMT3L and the R736H/DNMT3L complex was 3-fold more active than WT/DNMT3L. Similarly, formation of mixed R736H/DNMT3A WT FF-interfaces led to an increased activity. R736H/DNMT3L and mixed R736H/DNMT3A WT FF-interfaces were less stable than interfaces not involving R736H, suggesting that an increased flexibility of the mixed interfaces stimulates catalytic activity. Our data suggest that aberrant activity of DNMT3A R736H may lead to DNA hypermethylation in cancer cells which could cause changes in gene expression.

The lipidome of an omnivorous insect responds to diet composition and social environment.

Lipids are biomolecules with essential roles in metabolic processes, signaling, and cellular architecture. In this study, we investigated changes in the lipidome of the house cricket Acheta domesticus subjected to diets of different nutritional composition (i.e., protein to carbohydrate ratio) and two distinct social environments (i.e., solitary or in groups). We measured relative abundances of 811 lipid species in whole-body cricket samples using flow injection analysis coupled to tandem mass spectrometry. We assessed differences in the relative abundances of lipid species induced by diet composition and social environment in female and male A. domesticus. Additionally, we performed a functional analysis of the lipids with significant differences using a recently developed database. We found that most differences in the relative abundances of lipid species were explained by sex alone. Furthermore, the lipidome of female A. domesticus was responsive to diet composition. Females fed with the balanced diet had an even higher abundance of lipids involved in lipid storage than their counterparts fed with a protein-rich diet. Interestingly, the male cricket lipidome was not responsive to diet composition. In addition, the social environment did not induce significant changes in the lipid profile neither in female nor in male crickets.

New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1.

Patients who suffer from birch pollinosis often develop adverse reactions to the consumption of fresh apples due to the structural similarity of the allergens Bet v 1 and Mal d 1 from birch and apples, respectively. A different allergenic potential for Mal d 1 isoallergens is postulated, but approaches to quantify the Mal d 1 isoallergen-specific are missing. Therefore, a bottom-up proteomics approach was developed to quantify Mal d 1 by stable isotope dilution and microHPLC-QTOF analyses. Marker peptides for individual isoallergens (Mal d 1.01-Mal d 1.03 and Mal d 1.06), combinations thereof (Mal d 1.01 + 1.02, Mal d 1.02 + 1.06, and Mal d 1.04 + 1.05), and two global marker peptides, comprising Mal d 1.01 + 1.02 + 1.04 + 1.05 and Mal d 1.03 + 1.06 + 1.07 + 1.08 + 1.09, were identified. By the use of an extraction standard (r-Mal d 1_mut), an optimized protocol for extraction and tryptic digestion of apple proteins was developed, and the variety-specific extraction efficiency was monitored for the flesh and peel of apples. The Mal d 1 contents in flesh and peel of five commercial apple breeds and four apple varieties from orchard meadows were quantified isoallergen-specific.

Relative quantification of pork and beef in meat products using global and species-specific peptide markers for the authentication of meat composition.

We used global and species-specific peptide markers for a relative quantitative determination of pork and beef in raw and processed meat products made of the two meat species. Four groups of products were prepared (i.e., minced raw meats, sausages, raw and fried burgers) in order to represent products with different extents of food processing. In each group, the products varied in the pork/beef proportions. All products were analysed by multiple reaction monitoring mass spectrometry (MRM-MS) for the presence/concentration of pork- and beef-specific peptide markers, as well as global markers - peptides widely distributed in muscle tissue. The combined MRM-MS analysis of pork-specific peptide HPGDFGADAQGAMSK, beef-specific peptide VLGFHG and global marker LFDLR offered the most reliable validation of declared pork/beef compositions across the whole range of meat products. Our work suggests that a simultaneous analysis of global and species-specific peptide markers can be used for composition authentication in commercial pork/beef products.

Flanking sequences influence the activity of TET1 and TET2 methylcytosine dioxygenases and affect genomic 5hmC patterns.

TET dioxygenases convert 5-methylcytosine (5mC) preferentially in a CpG context into 5-hydroxymethylcytosine (5hmC) and higher oxidized forms, thereby initiating DNA demethylation, but details regarding the effects of the DNA sequences flanking the target 5mC site on TET activity are unknown. We investigated oxidation of libraries of DNA substrates containing one 5mC or 5hmC residue in randomized sequence context using single molecule readout of oxidation activity and sequence and show pronounced 20 and 70-fold flanking sequence effects on the catalytic activities of TET1 and TET2, respectively. Flanking sequence preferences were similar for TET1 and TET2 and also for 5mC and 5hmC substrates. Enhanced flanking sequence preferences were observed at non-CpG sites together with profound effects of flanking sequences on the specificity of TET2. TET flanking sequence preferences are reflected in genome-wide and local patterns of 5hmC and DNA demethylation in human and mouse cells indicating that they influence genomic DNA modification patterns in combination with locus specific targeting of TET enzymes.

Identification and Characterization of IgE-Reactive Proteins and a New Allergen (Cic a 1.01) from Chickpea (Cicer arietinum).

SCOPE: Chickpea (Cicer arietinum) allergy has frequently been reported particularly in Spain and India. Nevertheless, chickpea allergens are poorly characterized. The authors aim to identify and characterize potential allergens from chickpea. METHODS AND RESULTS: Candidate proteins are selected by an in silico approach or immunoglobuline E (IgE)-testing. Potential allergens are prepared as recombinant or natural proteins and characterized for structural integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD)-spectroscopy, and mass spectrometry (MS) analysis. IgE-sensitization pattern of Spanish chickpea allergic and German peanut and birch pollen sensitized patients are investigated using chickpea extracts and purified proteins. Chickpea allergic patients show individual and heterogeneous IgE-sensitization profiles with extracts from raw and boiled chickpeas. Chickpea proteins pathogenesis related protein family 10 (PR-10), a late embryogenesis abundant protein (LEA/DC-8), and a vicilin-containing fraction, but not 2S albumin, shows IgE reactivity with sera from chickpea, birch pollen, and peanut sensitized patients. Remarkably, allergenic vicilin, DC-8, and PR-10 are detected in the extract of boiled chickpeas. CONCLUSION: Several IgE-reactive chickpea allergens are identified. For the first time a yet not classified DC-8 protein is characterized as minor allergen (Cic a 1). Finally, the data suggest a potential risk for peanut allergic patients by IgE cross-reactivity with homologous chickpea proteins.

Diet composition and social environment determine food consumption, phenotype and fecundity in an omnivorous insect.

Nutrition is the single most important factor for individual's growth and reproduction. Consequently, the inability to reach the nutritional optimum imposes severe consequences for animal fitness. Yet, under natural conditions, organisms may face a mixture of stressors that can modulate the effects of nutritional asymmetry. For instance, stressful environments caused by intense interaction with conspecifics. Here, we subjected the house cricket Acheta domesticus to (i) either of two types of diet that have proved to affect cricket performance and (ii) simultaneously manipulated their social environment throughout their complete life cycle. We aimed to track sex-specific consequences for multiple traits during insect development throughout all life stages. Both factors affected critical life-history traits with potential population-level consequences: diet composition induced strong effects on insect development time, lifespan and fitness, while the social environment affected the number of nymphs that completed development, food consumption and whole-body lipid content. Additionally, both factors interactively determined female body mass. Our results highlight that insects may acquire and invest resources in a different manner when subjected to an intense interaction with conspecifics or when isolated. Furthermore, while only diet composition affected individual reproductive output, the social environment would determine the number of reproductive females, thus indirectly influencing population performance.

Mass Spectrometry-Based Untargeted Proteomics for the Assessment of Food Authenticity: The Case of Farmed Versus Wild-Type Salmon.

Background: Omics technologies have been widely applied in different fields, among which, proteomics is gaining increasing interest for its application to the authenticity of food products. MS, typically coupled with LC, represents a key technique for proteomics-related studies dedicated to fish and other seafood products by using a bottom-up approach. Objective and Methods: In this paper, the optimization of an untargeted proteomics-based method using LC separation and MS detection relying on a quadrupole time-of-flight mass spectrometer is described and applied to the analysis of Canadian farmed and wild-type salmon, followed by statistical analysis based on principal component (PC) analysis. Results and Conclusions: This untargeted approach, using a data-independent acquisition MS scheme, demonstrated the ability to effectively discriminate salmon belonging to the two classes. Furthermore, selected peptides showing high loadings on PC1 could represent potential candidate peptide markers able to discriminate farmed from wild-type salmon samples in the future.

German Government Official Methods Board Points the Way Forward: Launch of a New Working Group for Mass Spectrometry for Protein Analysis to Detect Food Fraud and Food Allergens.

The detection of food fraud and undeclared food allergens is one of the major challenges for competent authorities. Because adulterations are continuously adapted to the methods used to uncover them, the accomplishment of this task has become increasingly difficult over time. In recent years, various new promising methods for the detection of multiple food adulterants and multiple food allergens have been developed. Some of them utilize LC-MS to identify specific marker peptides. However, these methods have yet to be validated and standardized. For this reason, the German officials have established a working group with the objective of validating methods through multilaboratory validation studies. The experts of the working group also aim for the first time to standardize validated methods and to develop general validation criteria. This manuscript will highlight the current work of the group. For this purpose, an overview is given on the principles and applications of the new mass spectrometric methods. Moreover, requirements and the present work of other institutions regarding method validation are described.

Determination of food allergens by LC-MS: Impacts of sample preparation, food matrix, and thermal processing on peptide detectability and quantification.

Food allergies are a growing worldwide concern and the contamination of products with food allergens represents a significant health risk to allergic consumers. With the introduction of reference doses, quantitative methods are needed for the monitoring of allergen levels, and the potential of LC-MS/MS is of hugely growing interest. In this study, we demonstrate that relevant food matrices (bakery products and chocolates) and thermal food processing substantially influence the quantification of 18 marker peptides from various nut and peanut allergens via targeted proteomics. In addition, we characterize the individual release kinetics of marker peptides and provide examples for metastable marker peptide candidates. Matrix recovery rates overall ranged between 15 and 250% with the observed variation being linked to the individual peptide structure as well as to specific matrix interferences. In contrast, thermal processing considerably influences the detectability of allergens on the protein level as different marker peptides from the identical parent allergen are similarly affected, leading to a loss in signal of up to 83% in extreme cases after a 45-min simulated baking. Provided data are finally used for evaluation of different calibrators as well as the overall potential and challenges of LC-MS for the absolute quantification of food allergens. SIGNIFICANCE: With the scientific discussion moving towards a risk-based management of food allergens, including the establishment of threshold doses, robust methods for the absolute quantification of allergens in food samples are urgently needed. Because the currently used antibody- and DNA-based technologies show severe limitations in terms of specificity and reproducibility, LC-MS has emerged as a promising alternative. Its application to absolute quantification, however, first requires an understanding of the various impacts that affect quantification results, including different food matrices, sample preparation, and thermal processing of foodstuffs. Knowledge of these factors, which are assessed as part of a comprehensive survey in this study, is also an important prerequisite to evaluate means of calibration for an LC-MS-based quantification of food allergens.

Characterization and Detection of Food Allergens Using High-Resolution Mass Spectrometry: Current Status and Future Perspective.

Allergic reactions to food are among the major food safety concerns in industrialized countries, and it is estimated that approximately 5% of the population suffers from immunoglobulin-E-mediated food allergy. High-resolution mass spectrometry has become one of the most important techniques for the molecular characterization of allergens, including structural modification, degradation in the gastrointestinal environment, or identification of suitable marker peptides for the development of novel analytical approaches, in the past decade. This perspective aims to briefly summarize the current situation and discuss future developments.

Novel Approaches for the MS-Based Detection of Food Allergens: High Resolution, MS³, and Beyond.

The prevalence of allergic reactions to food is believed to be increasing in industrialized countries worldwide. One of the major tasks in risk management is, therefore, the analytical surveillance of allergen contamination in food and targeted proteomics using MS, which is of hugely growing interest due to its specificity and sensitivity and the possibility to analyze multiple allergens in parallel. Though approximately 200 different foods have been described as having the potential to elicit allergic reactions, current regional labeling requirements are focused on the 5-14 priority allergens that elicit the vast majority of severe reactions or that pose a risk as hidden allergens in food production. MS-based detection methods have been published for the majority of priority allergens, and this review provides an overview of the different methodological approaches, namely multiple-reaction monitoring-, high-resolution MS-, and triple-stage MS-based methods. In addition, requirements for the identification and validation of specific marker peptides and the influence of thermal processing and structural heterogeneity of allergens are discussed.

Gastrointestinal digestion of hazelnut allergens on molecular level: Elucidation of degradation kinetics and resistant immunoactive peptides using mass spectrometry.

SCOPE: Allergy to hazelnut seeds ranks among the most prevalent food allergies in Europe. The aim of this study was to elucidate the gastrointestinal digestion of hazelnut allergens on molecular level. METHODS AND RESULTS: Hazelnut flour was digested in vitro following the Infogest consensus model. For six allergenic proteins, the time-dependent course of digestion was monitored by SDS-PAGE and HPLC-MS/MS, and degradation products were characterized by a bottom-up proteomics approach. Depending on the molecular structure, a specific biochemical fate was observed for each allergen, and degradation kinetics were traced back to the peptide level. 1183 peptides were characterized, including 130 peptides that carry known IgE-binding epitopes and may represent sensitizers for hazelnut allergy. The kinetics of peptide formation and degradation were determined by label-free quantification and follow a complex multi-stage mechanism. CONCLUSION: We present a comprehensive survey on the gastrointestinal digestion of a relevant allergenic food on level of the peptidome, including the first systematic characterization and quantification of degradation products. This provides information on the differential resistance of plant food allergens and their structural elements undergoing digestion and forms the basis for a deeper understanding of the molecular principles responsible for sensitization to food allergy.

Structural Characterization of the Allergenic 2S Albumin Cor a 14: Comparing Proteoform Patterns across Hazelnut Cultivars.

The hazelnut allergen Cor a 14 belongs to the 2S albumins, a family of heterodimeric seed storage proteins exhibiting a high degree of structural diversity. Given its relevance as an allergen and the potential to elicit severe reactions, elucidation of the sequence heterogeneity of naturally occurring Cor a 14 is essential for the development of reliable diagnostics and risk evaluation. We therefore performed a comprehensive survey on the proteoforms of Cor a 14 and determined their quantitative distribution in three different hazelnut cultivars by a combinatory HPLC-HRMS approach including bottom-up and intact mass analysis. Compared with the Cor a 14 prototype sequence, we identified three sequence polymorphisms, two of the small and one of the large subunit, and elucidated their specific pairing on the protein level. Furthermore, we located a pronounced microheterogeneity on the protein termini and, for the first time, provide data on varying proteoform patterns between different cultivars of an allergenic seed. Together, these data present the basis for a more detailed investigation on the allergenicity of Cor a 14 in different cultivars and constitute, to be best of our knowledge, the largest set of proteoforms so far reported for a 2S albumin.

MRM3-based LC-MS multi-method for the detection and quantification of nut allergens.

Food allergies have become a global challenge to food safety in industrialized countries in recent years. With governmental monitoring and legislation moving towards the establishment of threshold allergen doses, there is a need for sensitive and quantitative analytical methods for the determination of allergenic food contaminants. Targeted proteomics employing liquid chromatography-mass spectrometry (LC-MS) has emerged as a promising technique that offers increased specificity and reproducibility compared to antibody and DNA-based technologies. As the detection of trace levels of allergenic food contaminants also demands excellent sensitivity, we aimed to significantly increase the analytical performance of LC-MS by utilizing multiple reaction monitoring cubed (MRM3) technology. Following a bottom-up proteomics approach, including a straightforward sample preparation process, 38 MRM3 experiments specific to 18 proteotypic peptides were developed and optimized. This permitted the highly specific identification of peanut, almond, cashew, hazelnut, pistachio, and walnut. The analytical performance of the method was assessed for three relevant food matrices with different chemical compositions. Limits of detection were around 1 µg/g or below in fortified matrix samples, not accounting for the effects of food processing. Compared to an MRM-based approach, the MRM3-based method showed an increase in sensitivity of up to 30-fold. Regression analysis demonstrated high linearity of the MRM3 signal in spiked matrix samples together with robust intersample reproducibility, confirming that the method is highly applicable for quantitative purposes. To the best of our knowledge, we describe here the most sensitive LC-MS multi-method for food allergen detection thus far. In addition, this is the first study that systematically compares MRM3 with MRM for the analysis of complex foods. Graphical abstract Allergen detection by MRM3.

New High-Performance Liquid Chromatography Coupled Mass Spectrometry Method for the Detection of Lobster and Shrimp Allergens in Food Samples via Multiple Reaction Monitoring and Multiple Reaction Monitoring Cubed.

Crustacean shellfish allergy ranks among the most frequent and severe food allergies for adults, demanding rugged and sensitive analytical routine methods. The objective of this study was therefore to develop a mass spectrometric approach for the detection of contamination with shrimp and lobster, two economically important types of crustaceans, in complex food matrices. Following a biomarker approach, we identified proteotypic peptides and developed a multiple reaction monitoring (MRM) method allowing for the identification and differentiation of shrimp and lobster in the food matrix at concentrations down to 0.1%. To further enhance sensitivity, we employed the MRM-cubed (MRM(3)) mode, which allowed us to detect crustaceans down to concentrations of 25 µg/g (crustacean/food, 0.0025%). We hereby present the first mass spectrometric method for the detection of shrimp and lobster in food matrices.

Comprehensive peptide marker identification for the detection of multiple nut allergens using a non-targeted LC-HRMS multi-method.

Food allergies have emerged as a global problem over the last few decades; therefore, reliable and sensitive analytical methods to ensure food safety for allergic consumers are required. The application of mass spectrometry is of growing interest in this field and several procedures based on low resolution tandem mass spectrometry using single tryptic peptides as analytical targets have recently been described. However, a comprehensive survey of marker peptides for the development of multi-methods is still missing, as is a consensus guide to marker identification. In this study, we therefore report a consistent approach to the development of liquid chromatography-mass spectrometry (LC-MS) multi-screening methods for the detection of allergens in food matrices. Proteotypic peptides were identified by a shotgun proteomics approach and verified through a thorough investigation of specificity and sensitivity. On the basis of this procedure, we identified 44 suitable tryptic marker peptides from six allergenic nut species and developed the first analytical LC-MS method for the detection of trace nut contaminations in processed foods using high resolution mass spectrometry (HRMS). The analysis of spiked matrix samples gave limits of detection (LODs) below 10 µg/g for several nuts; these LODs are comparable with routinely used methods such as ELISA and PCR. Notably, the HRMS approach can be used in an untargeted fashion to identify multiple allergens also retrospectively. In conclusion, we present here the so far largest consensus set of analytical markers from nut allergens and to the best of our knowledge the first multi-allergen method based on LC-HRMS.