Analysis of Allergen Proteins in Food Sample using LC-MS/MS

Significance of the topic

The accurate detection of food allergens is critical to protect allergic consumers from severe immune reactions. Traditional methods such as ELISA and PCR may suffer from cross-reactivity or lack of protein-level specificity. LC-MS/MS offers targeted peptide-based analysis, combining high specificity and multiplex capability for allergen monitoring in complex food matrices.

Objectives and overview of the study

This study aimed to develop and validate new multiple reaction monitoring (MRM) transitions for 16 food allergens, including eight mandatory allergens in Japan and additional nuts and fruits. Calibration curves were constructed, and the method’s specificity was assessed in processed food samples spiked at defined concentrations.

Methodology and sample preparation

• Protein extraction using proprietary allergen extraction reagents.
• Reduction of disulfide bonds with dithiothreitol and alkylation with iodoacetamide.
• Enzymatic digestion with trypsin aided by S-Trap cleanup.
• Removal of reagents via solid-phase extraction, concentration by centrifugal evaporation, and reconstitution.

Instrumentation

• Shimadzu Nexera X3 UHPLC system for peptide separation.
• Shimadzu LCMS-9030 Q-TOF mass spectrometer for data-dependent amino acid sequence confirmation.
• Shimadzu LCMS-8060RX triple quadrupole mass spectrometer for optimized MRM quantification.

Main results and discussion

• Specific MRM transitions were successfully established for four nut allergens and four fruit allergens, showing linear calibration (R2 >0.995) over 1–10 mg/kg concentration range.
• Cross-reactivity testing in processed chicken rice spiked with 16 allergens indicated minimal false positives, with interference rated on a four-tier scale; most peptides showed negligible background.
• Peptides from orange allergen exhibited interference and were classified as unacceptable, highlighting the need for alternative targets.

Benefits and practical applications of the method

• Simultaneous multi-allergen screening with high specificity reduces false positives from closely related proteins.
• Quantitative results based on total protein calibration align with regulatory labeling requirements.
• The approach complements or replaces ELISA and PCR in quality control laboratories for processed food analysis.

Future trends and potential applications

• Development of additional MRM transitions to cover challenging allergens such as orange proteins.
• Standardization of extraction and digestion protocols to facilitate method transfer and regulatory acceptance.
• Integration with high-throughput automation for routine allergen surveillance in the food industry.

Conclusion

The optimized LC-MS/MS workflow provides a robust platform for targeted quantification of multiple food allergens with excellent specificity and sensitivity. Continued method refinement and standardization will support broader implementation in food safety testing.

Reference

• Maeshima N, Tokami K, Inagaki E, Kobayashi M. Analysis of Allergen Proteins in Food Sample using LC-MS/MS. Shimadzu Corporation and Saika Technological Institute Foundation.