Selection of Peptide Targets for Species-independent Quantitation of Fish Allergens

Importance of the topic

Food allergens derived from fish pose significant risks to sensitive individuals, making reliable detection methods essential for consumer safety and regulatory compliance. The diverse range of fish species consumed worldwide complicates allergen quantitation, as conventional assays often fail to perform uniformly across different species.

Study objectives and overview

The study aimed to develop a species-independent workflow for quantifying major fish allergens by identifying peptide targets that are (1) shared across commonly consumed fish, (2) exhibit consistent abundance, and (3) are absent from non-fish sources. Major allergens—parvalbumin, enolase, and aldolase—were evaluated in nine fish species representing broad taxonomic groups.

Methodology

Sample preparation involved extracting proteins from fillet tissue of nine PCR-verified fish species (cod, pollock, herring, salmon, tuna, skate, tilapia, grouper, halibut) using a urea/thiourea reduction protocol. Each species was processed in three biological replicates.

Untargeted data-dependent acquisition was performed on a high-resolution Orbitrap platform. Peptide and protein identification, along with label-free quantitation, were conducted using de novo and database searches against UniProt sequences for each species. Quantitative data were further processed in Excel. Candidate peptides were screened for species specificity via BLAST P against the NCBI database.

Used instrumentation

• Thermo Scientific Q Exactive HF mass spectrometer
• Thermo Scientific UltiMate 3000 nanoRS UPLC
• Waters Acquity UPLC M-Class Peptide CSH C18 Column (1.7 µm, 75 µm × 250 mm)
• PeaksQ v8.5 software for identification and quantitation

Main results and discussion

• Parvalbumin: 51 peptides from 14 isoforms detected; only 4 isoform families common to all fish; abundance varied up to six-fold—indicating poor cross-species consistency.
• Enolase: 94 peptides from 6 isoforms; top3 quantitation revealed an 11-fold abundance range—highlighting variable expression across species.
• Aldolase: 63 peptides from 7 isoforms; predominant isoform quantified by top3, minor isoforms by single peptides; abundance varied ten-fold.
• Universal peptide candidates: Approximately 200 peptides with coefficient of variation below 150% across all species; BLAST screening narrowed these to ~25% fish-specific peptides, mainly derived from muscle proteins; only one peptide each from major and minor allergen families met all criteria.

Benefits and practical applications

The proposed DDA-based selection workflow provides a systematic route to discover peptide markers for multi-species allergen quantitation. Identified universal peptides can form the basis of targeted mass spectrometry assays (e.g., PRM) for more reliable detection than existing ELISA methods focused on parvalbumin.

Future trends and possibilities

• Validate candidate peptides using targeted PRM assays across individual fish species and in complex food matrices.
• Expand proteome databases for under-represented species to minimize selection bias.
• Apply the workflow to other cross-species allergen detection scenarios and to non-allergen food contaminants.

Conclusion

The study demonstrates that parvalbumin, enolase, and aldolase exhibit significant inter-species variability, limiting their utility as universal allergen markers. A DDA-driven peptide selection strategy successfully identified a small set of fish-specific, consistently abundant peptides suitable for species-independent quantitation, paving the way for robust mass spectrometry–based allergen assays.

References

• Marsh JT, Yang C, Johnson P. Selection of Peptide Targets for Species-independent Quantitation of Fish Allergens. Department of Food Science and Technology, University of Nebraska-Lincoln & Thermo Fisher Scientific.
• USDA National Institute of Food and Agriculture. Improving Food Safety Grant 1019035.