A Complete Discovery Workflow for Species- Specific Gelatin Identification

Significance of the Topic

Gelatin is a widely used ingredient in food, pharmaceutical, and cosmetic products. Accurate determination of its animal origin is critical for consumer safety, regulatory compliance, and adherence to religious dietary laws such as halal and kosher. Traditional authentication methods face limitations in sensitivity and species specificity, whereas proteomics-based LC-MS workflows offer robust identification of species-specific peptide markers.

Aims and Overview of the Study

This work presents a complete discovery workflow for the identification of bovine and porcine gelatin using proteomics. The objective is to develop a standardized and reproducible protocol that combines streamlined sample preparation, ultra-performance liquid chromatography, and data-independent acquisition mass spectrometry to discover and validate species-specific peptide markers.

Methodology and Instrumentation

• Sample Preparation: Gelatin standards were solubilized in ammonium bicarbonate, then processed using the ProteinWorks eXpress Digest Kit through a five-step protocol involving reduction, alkylation, and tryptic digestion.
• Chromatographic Separation: Peptide mixtures were separated on an ACQUITY Premier HSS T3 UPLC column (1.8 µm, 2.1 × 100 mm) using a water/acetonitrile gradient on the ACQUITY UPLC I-Class PLUS system.
• Mass Spectrometry: A Xevo G2-XS QTof mass spectrometer operated in MSE mode alternated between low and high collision energies to acquire both precursor and fragment ion data in a data-independent manner.
• Data Analysis: Progenesis QI for Proteomics performed alignment, normalization, and peak picking. Discriminatory features were selected via OPLS-DA in EZInfo, then identified using an Ion Accounting workflow against Sus scrofa and Bos taurus UniProt databases (FDR < 1%, carbamidomethylation of cysteines, oxidation of methionines).

Main Results and Discussion

Triplicate analyses of commercial bovine and porcine gelatin standards produced reproducible peptide profiles. An S-plot highlighted the most discriminating features between species. Example identification confirmed peptide SGDRGETGPAGPAGPIGPVGAR as porcine-specific. Principal component analysis and normalized abundance plots demonstrated clear separation of samples by species. A final list of signature peptides for bovine and porcine gelatin was assembled for future targeted assays.

Benefits and Practical Applications

• The ready-to-use ProteinWorks eXpress Digest Kit simplifies and standardizes sample preparation, reducing development time.
• MSE data acquisition provides comprehensive ion information without predefined inclusion lists.
• Identified peptide markers enable specific authentication of gelatin origin, supporting regulatory and religious compliance.

Used Instrumentation

• ACQUITY UPLC I-Class PLUS system
• ACQUITY Premier HSS T3 UPLC column (1.8 µm, 2.1 × 100 mm)
• Xevo G2-XS QTof mass spectrometer

Future Trends and Potential Applications

• Development of targeted MRM or PRM LC-MS/MS assays for routine quantitative authentication of gelatin in complex products.
• Expansion to mixed-species detection and low-level adulteration screening in food and pharmaceuticals.
• Integration with automated sample preparation and chemometric models for high-throughput quality control.

Conclusion

A robust proteomics discovery workflow combining kit-based digestion, UPLC separation, and MSE mass spectrometry has been demonstrated for species-specific gelatin identification. The approach yields reproducible peptide markers for bovine and porcine gelatin, paving the way for targeted quantitative methods in quality assurance and regulatory testing.

Reference

1. Uddin SMK et al. Halal and Kosher Gelatin: Applications as Well as Detection Approaches with Challenges and Prospects. Food Bioscience. 2021;41:101422.
2. Ali ME, Suleiman N. Eleven Shades of Food Integrity: A Halal Supply Chain Perspective. Trends in Food Science & Technology. 2018;71:216–224.
3. Hameed AM et al. A Review of Gelatin Source Authentication Methods. Tropical Life Sciences Research. 2018;29(2):213–227.
4. Rohman A et al. Analytical Methods for Analysis of Porcine Gelatin in Food and Pharmaceutical Products for Halal Authentication. Trends in Food Science & Technology. 2020;101:122–132.
5. Ishaq A et al. Analytical Approaches to Determine Gelatin Authenticity in Food Systems: A Review. LWT. 2020;121:108968.
6. Valetta M et al. Mass Spectrometry-Based Protein and Peptide Profiling for Food Fraud, Traceability, and Authenticity. Food Chemistry. 2021;365:130456.
7. Cheng XL et al. Identification of Five Gelatins by UPLC/QTof-MS Using Principal Component Analysis. Journal of Pharmaceutical and Biomedical Analysis. 2012;62:191–195.
8. Grundy H et al. Determination of Species Origin of Gelatine in Foods and Pharmaceuticals by Mass Spectrometry. Food Chemistry. 2016;190:276–284.
9. Sha MX et al. Identification of Three Mammalian Gelatins by Liquid Chromatography–High Resolution Mass Spectrometry. LWT. 2018;89:74–86.
10. Deng G et al. Animal Origin Identification of Gelatin-Based Products Using LC–MS Methods: A Mini Review. Reviews in Analytical Chemistry. 2020;9:260–271.