Comparative study of concatemer efficiency as an isotope-labelled internal standard for allergen quantification

Mass spectrometry-based methods coupled with stable isotope dilution have become effective and widely used methods for the detection and quantification of food allergens. Current methods target signature peptides resulting from proteolytic digestion of proteins of the allergenic ingredient. The choice of appropriate stable isotope-labelled internal standard is crucial, given the diversity of encountered food matrices which can affect sample preparation and analysis. We propose the use of concatemer, an artificial and stable isotope-labelled protein composed of several concatenated signature peptides as internal standard. With a comparative analysis of three matrices contaminated with four allergens (egg, milk, peanut, and hazelnut), the concatemer approach was found to offer advantages associated with the use of labelled proteins, ideal but unaffordable, and circumvent certain limitations of traditionally used synthetic peptides as internal standards. Although used in the proteomic field for more than a decade, concatemer strategy has not yet been applied for food analysis

Structural analysis of the interaction between human cytokine BMP-2 and the antagonist Noggin reveals molecular details of cell chondrogenesis inhibition.

peer reviewedBone morphogenetic proteins (BMPs) are secreted cytokines belonging to the transforming growth factor-β (TGF-β) superfamily. New therapeutic approaches based on BMP activity, particularly for cartilage and bone repair, have sparked considerable interest; however, a lack of understanding of their interaction pathways and the side effects associated with their use as biopharmaceuticals have dampened initial enthusiasm. Here, we used BMP-2 as a model system to gain further insight into both the relationship between structure and function in BMPs, and the principles that govern affinity for their cognate antagonist Noggin. We produced BMP- 2 and Noggin as inclusion bodies in Escherichia coli and developed simple and efficient protocols for preparing pure and homogeneous (in terms of size distribution) solutions of the native dimeric forms of the two proteins. The identity and integrity of the proteins were confirmed using mass spectrometry. Additionally, several in vitro cell-based assays, including enzymatic measurements, RT-qPCR and matrix staining, demonstrated their biological activity during cell chondrogenic and hypertrophic differentiation. Furthermore, we characterized the simple 1:1 non-covalent interaction between the two ligands (KDca. 0.4 nM) using bio-layer interferometry and solved the crystal structure of the complex using X-ray diffraction methods. We identified the residues and binding forces involved in the interaction between the two proteins. Finally, results obtained with the BMP-2 N102D mutant suggest that Noggin is remarkably flexible and able to accommodate major structural changes at the BMP-2 level. Altogether, our findings provide insights into BMP-2 activity and reveal the molecular details of its interaction with Noggin

High-resolution mass spectrometry-based selection of peanut peptide biomarkers considering food processing and market type variation

To protect allergic patients and guarantee correct food labeling, robust, specific and sensitive detection methods are urgently needed. Mass spectrometry (MS)-based methods could overcome the limitations of current detection techniques. The first step in the development of an MS-based method is the identification of biomarkers, which are, in the case of food allergens, peptides. Here, we implemented a strategy to identify the most salient peptide biomarkers in peanuts. Processed peanut matrices were prepared and analyzed using an untargeted approach via high-resolution MS. More than 300 identified peptides were further filtered using selection criteria to strengthen the analytical performance of a future, routine quantitative method. The resulting 16 peptides are robust to food processing, specific to peanuts, and satisfy sequence-based criteria. The aspect of multiple protein isoforms is also considered in the selection tree, an aspect that is essential for a quantitative method's robustness but seldom, if ever, considered

Estimating the health-care usage associated with osteoarthritis and rheumatoid arthritis in an older adult population in ireland

An ageing population leads to increasing prevalence of age-related chronic conditions that present challenges to the health-care services. Despite this, in countries including Ireland, little is known about the health-care impact of conditions such as osteoarthritis or rheumatoid arthritis amongst older adults. A series of count models are developed to investigate the incremental health-care usage of individuals with either osteoarthritis or rheumatoid arthritis on the use of general practitioners (GP) services, outpatients' services, accident and emergency visits and inpatient nights. Both types of arthritic conditions lead to increased usage of GP and outpatients' services but not other hospital services. Differences in entitlements to care, as captured by the presence of a medical card in Ireland, lead to different health-care usage among arthritis sufferers. Translating the additional utilization into cost suggests a combined incremental annual cost of both types of arthritis of a,not sign13.6 million. Osteoarthritis and rheumatoid arthritis present challenges to health-care services in the context of an ageing population. In the case of Ireland the burden falls predominantly on primary health-care and outpatient services. Within the context of changing health-care service provision in Ireland, the results of this study have implications for future planning of service delivery

Development and Validation of a Quantitative Method for Multiple Allergen Detection in Food Using Concatemer-Based Isotope Dilution Mass Spectrometry

Background Accurate food labeling is essential to protect allergic consumers. However, allergen contaminations may occur during the whole food production process. Reliable, sensitive, and robust methods for detecting multiple allergens in food are needed. Objective This work aims to develop and validate an LC coupled to tandem mass spectrometry (MS/MS) method for the detection and quantification of hazelnuts, peanuts, milk, and eggs in processed food products. Methods In-house-produced incurred test materials, cookies and chocolates, were used for the method development and validation. The quantification was based on the standard addition strategy using qualified reference materials as allergen protein standards and an innovative stable isotope-labeled concatemer as an internal standard. Results A method targeting 19 allergen-specific peptides was developed and validated in two laboratories, which strengthens its robustness. The AOAC INTERNATIONAL performance requirements for repeatability, intermediate precision, reproducibility, and recovery were reached for at least one peptide per allergen across both matrixes, and quantification limits complied with the action levels of the Food Industry Guide to the Voluntary Incidental Trace Allergen Labelling (VITAL (R)) Program Version 3.0. Conclusion The combination of incurred test materials, standard addition strategy, and stable isotope-labeled concatemer as an internal standard allowed us to develop and validate a robust method for detecting and quantifying multiple allergens in food with sufficient sensitivity to protect allergic consumers. Highlights: The combination of characterized incurred test material, calibration with certified reference material, a single stable isotope labelled concatemer and cross-lab validation result in the required standardization and harmonization in food allergen detection according to the stakeholders' group to assess the robustness of our method

Selecting processing robust markers using high resolution mass spectrometry for the detection of milk in food products

Background Cow's milk allergy is one of the most reported food allergies in Europe. To help patients suffering from food allergies it is important to be able to detect milk in different foods. An analytical method that is gaining interest in the field of allergen detection is ultrahigh performance liquid chromatography-tandem mass spectrometry, where the analyte is a target peptide. When these peptide biomarkers are selected, the effect of food processing should be taken into account to allow a robust detection method. Objective This work aims at identifying such processing stable peptide markers for milk for the ultrahigh performance liquid chromatography-tandem mass spectrometry based detection of food allergens in different food products. Method Milk-incurred food materials that underwent several processing techniques were produced. This was followed by establishing tryptic peptide profiles from each matrix using ultrahigh performance liquid chromatography-high resolution mass spectrometry. Results A careful comparison of peptide profiles/intensities and the use of specific exclusion criteria resulted in the selection of eight peptide biomarkers suitable for application in ultrahigh performance liquid chromatography-tandem mass spectrometry based milk detection methods. One of these markers is an alpha-lactalbumin specific peptide, which has been determined to be stable in different incurred materials for the first time. Conclusions To our knowledge, this is the first systematic and experimentally based approach for the selection of suitable milk peptide biomarkers robust toward multiple, often applied food processing techniques for milk. Ensuring the exact knowledge of the food processing circumstances by starting from well-defined raw material and using fully controlled settings to produce incurred test material allowed the construction of a peptide database with robust markers. These robust markers can be used for the development of a robust detection method for milk in different food matrixes