Cooked meat product analysis by LCMS/MS to determine between animal species using proteotypic peptide quantitative proteomic analysis

Importance of the topic

Analytical methods that accurately identify the animal species in cooked meat products are essential to ensure food authenticity and protect consumers from fraud or unintended contamination. Conventional DNA tests like PCR can be time-consuming, costly and prone to degradation in processed foods. Targeted proteomics using LC-MS/MS and proteotypic peptides offers a sensitive and specific alternative, even after heat treatment.

Study objectives and overview

This study aims to develop and validate a quantitative LC-MS/MS proteomic workflow to distinguish between beef, pork, chicken and horse in cooked and mixed meat products. Proteotypic peptides from myosin and type I collagen were selected to serve as species-specific markers and quantified using multiple reaction monitoring (MRM).

Methodology

Sample preparation included:

• Homogenisation of 150 mg meat in urea/thiourea buffer, followed by reduction (DTT) and alkylation (iodoacetamide).
• Tryptic digestion overnight and solid phase extraction cleanup with polymeric reversed-phase cartridges.
• Reconstitution in low organic mobile phase before LC-MS/MS analysis.

Peptide targets for each species were identified from Uniprot myosin and collagen databases and optimized in Skyline software. MRM transitions and collision energies were established to maximize specificity.

Used instrumentation

Liquid chromatography:

• Nexera UPLC system with Phenomenex Aeris C18 column (150×2.1 mm, 1.7 µm) at 50 °C, 0.3 mL/min flow, 2 µL injection.
• Gradient from 5% to 40% acetonitrile over 35 min, then to 100% before re-equilibration.

Mass spectrometry:

• Shimadzu LCMS-8050 with heated electrospray ionisation (ESI) and polarity switching.
• Interface at 400 °C, desolvation line at 150 °C, block at 250 °C, gas flows tuned for optimal desolvation.
• MRM dwell times of 47–117 ms and collision gas pressure at 350 kPa.

Key results and discussion

Heat treatment led to 19–52% weight loss across products, yet signal intensities of target peptides increased due to analyte concentration upon dehydration. Specific findings include:

• Beef myosin peptide intensity rose despite 29% weight loss after cooking.
• Type I collagen peptide for pork was readily detected in mixed pastes and sausages.
• Horse myosin peptides were distinguishable from other species and detected at 1% spiked levels in beef.

These results underscore the method’s robustness to processing and capability to quantify low-level adulteration.

Benefits and practical applications of the method

• Rapid and sensitive detection of species-specific peptides in cooked and processed meats.
• High specificity reduces cross-reactivity and false positives.
• Quantitative capability enables estimation of contaminant levels.
• Complementary to PCR workflows, offering tissue-level information.

Future trends and potential applications

Advancements may include:

• Multiplexed assays covering a broader range of species and tissue markers.
• Automation of sample preparation and data analysis for high-throughput testing.
• Integration with regulatory monitoring and quality control in food supply chains.
• Expansion to allergen and toxin profiling using targeted proteomics.

Conclusion

This study demonstrates that targeted LC-MS/MS proteomic analysis of proteotypic peptides is a powerful tool for species identification in cooked meat products. The approach offers high sensitivity, specificity and quantification capability, addressing limitations of DNA-based methods, and supports more comprehensive food authenticity testing.

Reference

• Montowska M et al. Anal Chem. 2014;86:10257–10265.
• Van der Breggen C et al. J Agric Food Chem. 2013;61:11986–11994.
• Van der Breggen C et al. J Agric Food Chem. 2014;62:9428–9435.
• Zhang G et al. Food Hydrocolloids. 2009;23:2001–2007.