IMSC: Ultra-Fast Analysis of Allergens Using Capillary Electrophoresis Coupled to Mass Spectrometry and Ultra Violet Photodissociation

Importance of the Topic

Food allergies pose significant risks to sensitized individuals and demand reliable detection methods. Fish allergens, particularly parvalbumins, are heat-stable proteins that trigger allergic reactions. Rapid identification and authentication of allergenic proteins and fish species are critical for food safety and regulatory compliance.

Objectives and Study Overview

This study aimed to develop a simple, fast workflow combining capillary electrophoresis with top-down mass spectrometry to detect fish allergens and authenticate species. The method isolates thermostable proteins, separates isoforms by CE, and characterizes them using high-resolution MS.

Methodology and Instrumentation

Protein Extraction and Cleanup:

• Mechanical homogenization of 1 g muscle tissue from commercial fish species
• Heat treatment at 70 °C for 5 min to precipitate non-thermostable proteins
• Stage-tip cleanup of soluble fraction

CE Separation and Ionization:

• Agilent 7100 CE with cation-coated (–30 kV) and neutral-coated (+30 kV) capillaries (100 cm × 50 μm)
• Methanol/water/formic acid sheath liquid with electro-osmotic flow (CMP/EMASS-II ion source on 3D-printed stage)

Mass Spectrometry:

• Thermo Orbitrap Fusion Lumos Tribrid MS with UVPD source
• MS resolution at 120 k (m/z 200)
• Fragmentation modes: HCD, ETD, EThcD, UVPD

Data Analysis:

• Thermo Xcalibur 4.0, QualBrowser
• ProSight Lite for top-down protein identification

Main Results and Discussion

Thermostable sarcoplasmic proteins were enriched, revealing β-parvalbumins (~11 kDa) as major allergens. CE separation resolved multiple β1 and β2 isoforms, generating distinct migration profiles. Top-down MS achieved mass accuracy within 1.8–4.1 ppm. Protein sequence coverage varied by fragmentation strategy, reaching up to 81 % with EThcD. Two Hake species (Merluccius paradoxus, M. merluccius) were distinguished by their parvalbumin isoform profiles.

Benefits and Practical Applications

• Rapid, high-specificity detection of fish allergens in complex food matrices
• Simultaneous species authentication via isoform differentiation
• Compatibility with CE-chip formats for on-site testing

Future Trends and Applications

Emerging miniaturized CE-MS platforms could enable point-of-care allergen testing. Expanded use of UVPD and hybrid fragmentation will improve coverage and throughput. Broader application to other allergens and integration with lab-on-chip devices are anticipated.

Conclusion

The presented CE-MS/MS top-down workflow offers a reliable, fast approach for fish allergen identification and species authenticity. High-resolution separation and diverse fragmentation strategies enable robust detection of parvalbumin isoforms, supporting food safety initiatives.

Instrumentation

• Agilent 7100 Capillary Electrophoresis system
• Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer
• UVPD source and EThcD/HCD/ETD fragmentation
• 3D-printed CMP/EMASS-II sheath-flow ion source

References

1. Carrera M.; Canas B.; Gallardo J.M. Journal of Proteomics, 2012, 75, 3211–3220
2. CMP Scientific, Brooklyn, NY (ion source)
3. Alcor Bioseparations LLC, Palo Alto, CA (CE interfaces)