LC-HRMS Multi-Allergen Detection of Nuts Contamination

The incidence of allergy to food components is on the rise, meaning that producers and public health officials must be vigilant in ensuring absence or comprehensive declaration of potential allergens on food labels. Korte et al. (2016) present a novel high-resolution mass spectrometric (HRMS) method for detecting multiple nut allergens in a food matrix¹.

Trace contamination with food allergens can be life-threatening to highly allergic individuals, which is why food producers must be comprehensive in their declarations on labels. Food safety officials must also be comprehensive in their testing for potential risks. Both parties require sensitivity and accuracy in testing methods. Current methodologies for testing for the presence of nut allergens are very sensitive; however, immunological methods such as enzyme-linked immunosorbent assay (ELISA) can yield false positives through cross-reactivity with matrix since many allergen epitopes are not fully characterized. In addition, molecular methods such as polymerase chain reaction (PCR) do not detect the allergenic proteins, only the DNA. Neither of these methods are suitable for multiplexing, meaning that for full characterization, many different tests are required for each food product.

Korte et al. propose the harness the power and sensitivity of HRMS to multiplex nut allergen detection. Using a non-targeted approach on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific), the team developed a screening method that accurately reports the presence of contamination by identifying multiple peptide markers for various nut species-specific allergens.k

Almond Prunus dulcis	Cashew Anacardium occidentale
Hazelnut Corylus avellana	Peanut Arachis lypogaea
Pistachio Pistacia vera	Walnut Juglans regia

Table 1: nut species tested

The research team obtained commercial samples of the nuts shown in Table 1, extracted the proteins using standard methodologies and then separated the proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). They precipitated the proteins obtained in 20% trichloroacetic acid then digested them with trypsin. Kork et al. analyzed the peptide digests by LC-HRMS on an Accela high-performance liquid chromatography (HPLC) system coupled with an LTQ Orbitrap XL hybrid ion trap-Orbitrap mass spectrometer (both Thermo Scientific). Operating the mass spectrometer in data-dependent acquisition mode (DDA), the team gathered spectral data to identify marker peptides by searching against the relevant UniProt databases.

Following buffer validation, the team found that extraction proceeded most effectively in a Tris buffer with 2M NaCl at pH9. They then proceeded with bottom-up MS analysis to select tryptic markers for each nut species. From the initial runs, the researchers selected 39 allergenic proteins across all six species, represented by 807 peptides in total. With filtering to select unique markers with no inter-species crossover, Korte et al. developed a set of 44 peptide markers representative of the nut species under examination. These markers represented proteins from the 2S, 7S, and 11S seed storage protein family in addition to 11S legume proteins.

The researchers conducted further validation steps with spiking studies to confirm that the markers did not show interference from food matrices (milk chocolate, bread, breakfast cereal). The team also conducted serial spiking studies in vanilla ice cream at nut protein concentrations of 9, 7, 5, 3 and 1µg/g to demonstrate that the approach was sufficiently sensitive (limits of detection, LODs below 10µg/g; coefficient of variation for triplicate testing at 2-16%) for food safety testing.

Korte et al. that LC-HRMS is a valid and sensitive method for multiplexed nut allergen detection in food matrices, demonstrating the ease of use, reliability and with reduced potential for interference. During the validation, the researchers found that the method was sufficiently sensitive to detect nut-on-nut contamination, showing mixing occurred during processing. Their panel of 44 markers for the six nut species includes at least one identifier for each nut. The team notes that some isoforms do occur and that these may exist at different levels dependent on the source. For this reason, they strongly support using at least three markers per species for accurate multiplexed nut allergen detection.

For further discussion on detection of allergens in food products visit our food and beverage learning center.

Reference

1. Korte, R. et al. (2016) “Comprehensive peptide marker identification for the detection of multiple nut allergens using a non-targeted LC–HRMS multi-method“, Analytical and Bioanalytical Chemistry 408 (pp.3059–69) DOI 10.1007/s00216-016-9384-4