IMSC: From Ocean To Table: An Integrated Mass Spectrometry Approach To Identify The Fish OnYour Plate

Significance of the Topic

Accurate identification of commercial fish species is critical to prevent fraud, ensure consumer safety and confidence, and support sustainable fisheries management. Proteomic techniques offer rapid, reliable tools for authenticating seafood products, addressing global challenges of resource overexploitation and mislabeling.

Objectives and Overview of the Study

This work presents an integrated mass spectrometry–based workflow combining bottom-up and top-down proteomic approaches for untargeted, label-free authentication of a commercial hake filet. The method aims to discriminate among closely related Merlucciidae species in under 30 minutes, leveraging intact mass and peptide biomarkers.

Methodology and Instrumentation

• Sample Preparation: 1 g of fish muscle homogenized in water, centrifuged, heat-treated at 70 °C for 5 min, and centrifuged again to isolate thermostable proteins.
• Bottom-Up Proteomics: Trypsin digestion (3 min, high-intensity ultrasound), desalting (Pierce™ Micro-Spin Columns), LC-MS/MS on Thermo Scientific™ Easy-nLC 1200 coupled to Q Exactive™ Hybrid Quadrupole-Orbitrap™, peptide separation on EASY-Spray™ column, data processed in Proteome Discoverer™ 2.1 against a composite Uniprot fish database.
• Top-Down Proteomics: Direct infusion of undigested supernatant into Q Exactive, MS and HCD MS/MS at high resolution, data deconvoluted in ProSight Lite.
• Targeted PRM: Development of a decision tree for parvalbumin-derived tryptic peptides to systematically discriminate Merlucciidae species.

Main Results and Discussion

• Bottom-Up Findings: Over 200 proteins identified; top hits were parvalbumin beta isoforms from M. merluccius, M. hubbsi, and M. paradoxus with >65 % sequence coverage but high homology impeded specific species assignment.
• Intact Mass Analysis: A dominant ~11.365 kDa mass corresponding to parvalbumin beta 2 allowed discrimination of M. paradoxus by characteristic mass shifts, with MS/MS confirming 45 % sequence coverage.
• PRM Decision Tree: Targeted selection of unique peptides (AEGTFK, SPADIK, SPAADIK) enabled unambiguous classification of Merlucciidae species despite their conserved proteomes.

Benefits and Practical Applications of the Method

• Speed: Complete authentication in less than 30 minutes from tissue to result.
• Sensitivity and Specificity: Combines untargeted discovery with targeted verification to resolve closely related species.
• Versatility: Applicable to routine quality control in seafood processing and regulatory enforcement.

Future Trends and Potential Applications

Integration of advanced feature detection algorithms (e.g., Minora Feature Detector, RT-Aligner, Feature Mapper) in Proteome Discoverer™ 2.2 will enhance label-free quantification capability and throughput. Expansion of targeted PRM panels and machine-learning–driven spectral libraries can further improve discrimination of diverse fish species and other food products.

Conclusion

The presented proteomic workflow demonstrates rapid, robust authentication of commercial hake samples, overcoming challenges posed by protein homology. Coupling intact mass profiling with targeted peptide markers provides a powerful approach for seafood species verification and fraud prevention.

Reference

1. Carrera M., Canas B., Lopez-Ferrer D. et al. Analytical Chemistry 2011, 83, 5688–5695.