Mass Spectrometry Based Allergen Detection: Applicability of Published Peptide Sequences for the Quantification of Different Peanut Varieties and Processed Peanuts Using Triple Quadrupole LC/MS

Significance of the Topic

Food allergies pose a serious health risk and avoiding allergenic ingredients remains the only reliable preventive strategy. Peanut allergy is one of the most common and severe food allergies. Accurate detection of peanut residues in food matrices is critical for consumer safety, regulatory compliance and quality control. Traditional methods such as ELISA and PCR can suffer from variable recoveries due to cultivar differences and food processing. Mass spectrometry (MS) offers a promising alternative with specific peptide markers that may be less sensitive to processing effects.

Objectives and Study Overview

This study evaluates whether published tryptic peptide sequences for the major peanut allergens Ara h1 and Ara h3 are applicable across diverse peanut varieties and processing conditions. Fourteen peanut samples—including raw, roasted, salted and peanut cream materials—were analyzed by triple quadrupole LC/MS and compared with ELISA measurements to assess assay robustness and quantitation variability.

Methodology and Instrumentation

Sample preparation involved cryogenic grinding, protein extraction, quantification, reduction, alkylation and overnight trypsin digestion. Peptide extracts were analyzed on an Agilent 1290 Infinity UHPLC coupled to an Agilent 6490 Triple Quadrupole MS with Jet Stream electrospray ionization. Dynamic multiple reaction monitoring (dMRM) was employed, using five to seven transitions per peptide. For comparison, the AgraQuant® F.A.S.T. Peanut ELISA kit was used following the manufacturer’s protocol.

Key Findings and Discussion

• Six peptide markers (three for Ara h1 and three for Ara h3) were reliably detected in all unprocessed peanut varieties, but signal intensities varied up to twofold between cultivars.
• Roasting caused a greater reduction in Ara h1 peptide signals (up to 14-fold) compared to Ara h3 (approximately 6-fold), indicating differential stability of allergen proteins under heat.
• ELISA results showed up to twofold variability among raw samples and up to eightfold signal reduction after roasting, broadly aligning with MS-based findings.

Benefits and Practical Applications

MS-based detection using selected peptide markers demonstrates comparable resilience to processing-induced losses as ELISA, with the added advantages of high specificity, multiplex capability and reduced cross-reactivity. This approach can improve accuracy of allergen quantification in complex or processed food matrices and support regulatory compliance and risk assessment in food manufacturing.

Future Trends and Opportunities

Advancements may include:

• Extension to additional allergenic proteins and food matrices.
• Integration of high-resolution MS and data-independent acquisition for broader peptide coverage.
• Development of multiplex panels for simultaneous detection of multiple allergens.
• Standardization of peptide markers across laboratories to enhance method reproducibility.

Conclusion

Published peptide sequences for Ara h1 and Ara h3 are suitable for quantitative MS-based detection across diverse peanut varieties. While processing effects reduce recoveries, MS approaches match ELISA in robustness and offer greater specificity. Implementation of this method can strengthen food allergen monitoring and protect allergic consumers.

References

1. Heick, J., et al. JAOAC 2011, 94(4), 1-9.