Targeted Proteomics Approach for Sensitive Detection of Bovine and Porcine Gelatins in Food, Pharmaceutical Capsules and Personal Care Products

Significance of the Topic

Gelatin, derived from bovine and porcine sources, is widely used in food, pharmaceutical, and personal care products. Reliable species identification is essential to meet religious, dietary, and labeling requirements due to high sequence homology between bovine and porcine gelatins.

Objectives and Study Overview

This work aimed to develop a targeted proteomics approach for sensitive detection and differentiation of bovine and porcine gelatin peptides using multiple reaction monitoring (MRM) on a triple-quadrupole LC–MS/MS platform (LCMS-8060). The method was applied to reference materials and commercial samples.

Methodology and Instrumentation

Sample Preparation and Digestion
• 5 mg gelatin dissolved in ammonium bicarbonate buffer, vortexed and centrifuged.
• Trypsin digestion at 37 °C overnight (enzyme:sample 1:100).
• Supernatant collected by centrifugation for LC–MS/MS analysis.

MRM Method Development
• In silico prediction of species-specific peptide candidates using UniProt and Skyline.
• Optimization of MRM transitions and collision energies based on reference digests.

Instrumentation

• Shimadzu LCMS-8060 triple-quadrupole MS/MS system
• Chromatography: Aeris peptide XB-C18 column (150 × 2.1 mm, 1.7 µm, 100 Å)
• Mobile phase A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile; gradient 5–50% B over 16 min; flow rate 0.3 mL/min; column temperature 40 °C
• ESI source: interface 300 °C; block 400 °C; desolvation line 250 °C; nebulizing gas N₂ 3 L/min; drying gas N₂ 10 L/min; heating gas (zero air) 10 L/min
• MRM acquisition in positive ion mode

Key Results and Discussion

• Nine bovine-specific and eight porcine-specific peptide markers were identified from collagen α-chains and validated experimentally.
• Proline hydroxylation can produce isobaric interferences, necessitating careful transition selection and sequence verification.
• All markers showed good repeatability (RSD < 15%) and allowed detection of 0.1% gelatin adulteration in spiked matrices.
• Screening of 19 commercial products revealed detection results consistent with labeling in most cases; some samples contained both bovine and porcine gelatins unexpectedly.

Benefits and Practical Applications of the Method

• Enables reliable species-origin verification for regulatory and religious compliance (Halal, Kosher).
• Suitable for complex matrices in food, pharmaceuticals, and cosmetics.
• High sensitivity and specificity support quality control and labeling accuracy.

Future Trends and Potential Applications

• Extension to other animal-derived ingredients and multi‐species detection strategies.
• Incorporation of high-resolution MS for expanded proteome coverage and discovery of additional markers.
• Automation of sample preparation and data processing for high-throughput routine analysis.

Conclusion

The developed MRM-based LCMS-8060 method delivers a robust, sensitive, and specific approach to distinguish bovine and porcine gelatins at trace levels (0.1%), facilitating accurate product labeling and adherence to dietary and religious standards.

References

• Grundy HH et al. Food Chem. 2016;190:276–284.
• Ali ME et al. Crit Rev Food Sci Nutr. 2016;1–17.
• Jumhawan U et al. Highly-Sensitive Detection of Multiple Porcine-Specific Peptides in Processed Foods by LC/MS/MS Method. Application News AD-0153.
• Nemati M et al. J Pharm Biomed Anal. 2004;34:485–492.
• Zhang G et al. Food Hydrocolloids. 2009;23:2001–2007.
• Cheng X et al. J Pharm Biomed Anal. 2012;62:191–195.