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Food Allergen Detection in Complex Matrices: Utilizing the Power of Mass Spectrometry

Team TFS
Team TFS
allergenThe Food Allergy Research and Resource Program (FARRP) is a food industry-oriented research and outreach program within the Department of Food Science and Technology at the University of Nebraska-Lincoln. FARRP was established in 1995 with the mission of providing solutions and assistance on food allergy issues to the food manufacturing industry, consumers, physicians, governmental agencies, and other stakeholders, through research, training and education, technical assistance, and information resources. FARRP currently includes more than 100 member companies that help support our research and outreach activities and that benefit from our services. Over its history of more than 20 years, FARRP has conducted research on a number of areas critical to food allergen management, including:

  • Development and improvement of methods for the detection of allergenic residues in foods

  • Evaluation of the efficacy of methods for the removal of allergenic residues from surfaces

  • Identification and characterization of allergenic proteins in foods

  • Development of approaches to assess the potential allergenicity of novel food protein sources

  • Establishment and validation of population threshold doses for allergenic foods

  • Assessment of the allergenicity of ingredients derived from allergenic sources

  • Evaluation of allergen identification and labeling (especially precautionary labeling)

Learn more about food allergens testing here.

I provided some insights into our work for the team:

What does your current work involve and what does this work bring to FARRP?

Much of my team’s research is currently focused on the development of mass spectrometry-based detection and quantification methods for food allergens. While immunoassay methods are by far the most common food allergen detection strategy and perform well in many applications, FARRP’s research over the years has identified gaps where immunoassays may not adequately detect food allergen residues. In particular, foods that have been extensively thermally processed (e.g. retorted, deep-fried, UHT processed) or subjected to fermentation or hydrolysis processes can be very difficult, if not impossible, to quantitatively detect with current immunoassays. Additionally, the detection of allergen-derived ingredients in which the proteins have been fractionated or modified can pose a number of challenges. Because MS methods do not depend on protein conformation and multiple peptides can be targeted for detection, methods can be developed to improve quantification of food allergens in these scenarios. Obtaining accurate quantitative information is critically important for evaluating health risks associated with food allergens, including through quantitative risk assessment (QRA) strategies that are employed by FARRP. In addition to improving detection and quantification, MS methods also serve as confirmatory methods when immunoassay or PCR methods deliver conflicting or unexpected results.

What is your experience with Orbitrap technology for non-targeted workflow and qualitative tool?  How do you prove the signal from a targeted method is the allergen of interest?

When we are working on developing a new targeted method, we often need to find out more about what proteins are present in a range of allergen-derived ingredients so that the methods can detect allergen presence regardless of the source. In order to do that, we use bottom-up proteomics techniques, which include identification based on comparisons between spectral data and protein sequence databases. Having information about which proteins are readily and reliably detectable in allergen-derived ingredients is critical for selecting robust targets for detection methods.

In the development of our methods, we use multiple tools to make sure our selected peptide targets are specific to the allergenic food of interest. Because many foods are closely related and have poorly described proteomes, we conduct both in silico analysis and laboratory screening of background food matrices to verify peptide specificity.

When we are analyzing a sample with a targeted method, we utilize a number of different pre-defined metrics to verify that the detected signals are from the target peptide of interest, including that the detected peptide is within a pre-set retention time window based on positive control analysis (accomplished utilizing scheduled PRM methods). At least three pre-determined product ions must be found, and product ion ratios must be consistent with spectral libraries or internal standards. In certain select cases with specific background ion interferences, we have also set product ion mass tolerances (5-10 ppm).

Do you feel that Orbitrap technology can help food testing companies identify the contamination and what kind of barriers do you see that will limit the use of this technology?

Food allergen analysis can pose a number of challenges, particularly for service laboratories, as they are commonly asked to analyze incredibly diverse food and ingredient matrices for the presence of a variety of undeclared food allergens, often with little information about the matrix composition. As it is not practically feasible to validate methods in every variation of food product, HRAM instruments such as Orbitrap may provide important advantages for the specificity of MS methods when applied across such a range of samples.

Unlike with immunoassays or small-molecule MS techniques, targeted proteomics methods, especially for food allergens in diverse matrices, require lengthy and comparatively costly sample preparation. Improvements to sample preparation workflows to better meet industry turnaround expectations could go a long way to increase adoption. In addition, current proteomics data analysis (even for targeted methods) requires a substantial amount of expertise and manual interpretation. Automated data analysis workflows and templates that reduce requirements for user expertise could make these approaches more widely implemented.

Looking to the future what do you think other scientist in food safety labs need to know about LC-HRAM MS?

Like any analytical method or instrument, HRAM MS is not a magical solution for food allergen analysis challenges. However, with the correct method design and validation this type of MS technology can be used to overcome a number of hurdles encountered in food allergen analysis. And while some specialized education and training may be required to implement targeted or non-targeted HRAM proteomics methods in a laboratory, it is not as large of a transition as it may seem.

Would you recommend this Orbitrap technology to other scientists looking into allergens for Food Safety not just for import but also export?

Q Exactive Orbitrap instruments in particular may be useful to other scientists interested in pursuing MS-based food allergen analysis, as they enable users to conduct both discovery and targeted proteomics experiments. While most food allergen analysis is likely to continue to use targeted methods, new analytical issues are continually arising for food allergens, and in some instances discovery proteomics strategies are critical to determining method specificity and evaluating health risks.