A Tough Nut to Crack: detection of peanuts in nuts with HRMS

As Monaci et al (2015) explain, when peanuts are packaged or stored adjacent to other nuts there is a risk of cross contamination. Since peanuts can cause serious allergic reactions even in small amounts, it is vital to accurately identify and isolate these sources of contamination. To this end, researchers have developed specialized testing such as ELISA based assays, which can detect the allergenic proteins in peanuts and other foods delivering results in around 60 minutes.

Mass spectrometry is another option utlized for detecting allergens in foods. This method is particularly useful for analyzing trace amounts of proteins, or when analyzing complex samples such as mixtures of nuts. For this reason, Monaci et al. chose to use liquid chromatography and mass spectrometry (LC/MS) to identify and quantify peanuts in mixtures of other nuts.

The researchers analyzed a mixture of hazelnuts, pistachios, almonds and walnuts that they ground into a powder. To this powder, they added peanuts at different amounts  down to 1,000µg per gram. The team then extracted, digested, and separated proteins. Using high performance liquid chromatography and mass spectrometry, (HPLC­-MS/MS) on a Velos Pro linear ion trap mass spectrometer, (Thermo Scientific) the team looked for peanut  markers. They used Proteome Discoverer software (version 1.4, Thermo Scientific) to analyze the raw data. Along with this, they also queried a customized food allergen database built in-­house that contained approximately 2800 sequences imported from UniProt KB.

Focusing on fragmentation patterns in the MS/MS spectra, the team looked for peptides with the highest Xcorr value. The Xcorr refers to the number of fragment ions common to two different peptides with the same precursor mass. As a result, the researchers identified six peptides abbreviated as: VYD, WLG, FNL, SPD, DLA, and GTG.

Next the researchers again fortified nut mixtures with peanuts and performed a high resolution mass spectrometry analysis (HRMS) using a benchtop high ­resolution Exactive mass spectrometer (Thermo Scientific), this time to look for the six potential peanut markers. For the HRMS analysis of the nut mixtures, the team used an HPLC pump equipped with an Accela autosampler (Thermo Scientific) equipped with an HESI II interface.

Monaci and colleagues excluded two of the original six possible peanut markers, since they were only detectable at the highest levels of peanuts. The researchers noted that the remaining four peptides were good qualitative markers based on calibration curves. Peptides VYD and WLG had the highest sensitivity and were good quantitative markers with a limit of detection as low as 4 µg of peanut proteins or 26 µg of peanuts in 1 g of matrix.

While mass spectrometry has been used in the past to look for peanut markers in other foods, this is the first application of high­-resolution Orbitrap ­based mass spectrometry specifically to identify peanuts in a mixture of other nuts. The advantage of this approach against traditional ELISA based methods is the possibility to detect the presence of peanut proteins in the sample with high confidence.

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Reference

Monaci L. et al. (2015) “High­resolution Orbitrap™­based mass spectrometry for rapid detection of peanuts in nuts.” Food Additives and Contaminants Part A Chemical Analysis Control Exposure & Risk Assessment 2015;32(10):1607­16. doi: 10.1080/19440049.2015.1070235. Epub 2015 Jul 29.