Final Report Summary - AUTHENTIMEAT (Authentication of meat products using ambient surface mass spectrometry) AUTHENTIMEAT intended to explore for the first time the potential of ambient surface mass spectrometry; including desorption electrospray ionization (DESI) and liquid extraction surface analysis (LESA) mass spectrometry, for authenticating meat products and developing a novel and fast method of authentication of processed meat products based on proteins and unique peptides. Methods of food adulteration are becoming increasingly refined, that is why the fight with adulteration and unlawful food product supplementation must be based on the application of new, fast and effective instrumental/analytical techniques that allow rigorous analysis of complex and processed products.The AUTHENTIMEAT proposes the use of ambient mass spectrometry techniques because of their ability for rapid detection of compounds directly from biological surfaces, and therefore because of their potential for high-throughput screening of meat products with minimal sample preparation. See Attachment 1 for workflow for DESI and LESA-MS experiments. Initially, standard proteins and mixtures up to five proteins (myoglobin, troponin C, actin, BSA, tropomyosin) were investigated by on-surface tryptic digest peptide mass fingerprinting analysis using DESI-MS and LESA-MS. Subsequently, peptidomic analysis of whole meat digests, dried, desorbed from a surface was carried out using tandem mass spectrometry and multivariate data analysis (MVA). Finally, LESA-MS/MS methodology was applied for rapid detection of heat stable peptide markers for five meat species, such as cattle, pig, horse, chicken and turkey, as well as validated on processed meat products. The results demonstrate that ambient ionization techniques such as DESI-MS and LESA-MS have great potential for species-specific analysis and differentiation of skeletal muscle proteins. However, LESA-MS gave a more reproducible analysis and greater sensitivity compared with DESI-MS. Raw and cooked meat digests were clearly discriminated and the most abundant skeletal muscle proteins were correctly classified according to the species. A number of 23 heat stable peptide markers unique to species and muscle protein were identified. Surface extraction and direct ambient MS analysis of mixtures of cooked meat species enabled detection of 10% (w/w) of pork, horse, and turkey meat, and 5% (w/w) of chicken meat in beef, using the developed LESA-MS/MS analysis. Average mass spectra obtained from in-solution digestion of meat mixtures and excellent differentiation between the mixtures using multivariate data analysis are presented in figure in Attachment 2. The developed methodology was also tested on 21 various processed meat products of unknown and known composition, including sausages, frankfurters, hot dogs, meat spreads. The study shows that ambient LESA-MS methodology displays specificity sufficient to be implemented effectively for the analysis of processed and complex peptide digests. The proposed approach is much faster and simpler than other measurement tools for meat speciation; it has potential for application in other areas of meat science or food production.The ability for a rapid analysis is a crucial issue in the case of processed and perishable food products. Protein composition of a product may vary depending not only on species but also on many other factors, including tissue type, storage time of the product and technological processes employed to manufacture it. Authentication is tough in complex processed products treated with high temperature and pressure, or subjected to drying, smoking or pH changes, because both proteins and DNA undergo considerable degradation. In this work, we have proof of principle that rapid and easy to use ambient methodology has the potential to be applied to routine analysis of complex and processed and meat products. Time of analysis could be shortened to approximately 1 h by the application of microwaves or ultrasonication to reduce the time of digestion process; at present a critical step for the duration of the analysis. This is a big advantage over the more time-consuming protocols based on electrophoresis or liquid chromatography. In this approach, the complex peptide mixture was directly analyzed without any pre-treatment or fractionation. The method has good specificity, since it enabled detection of more unique peptides and among them were some of the same peptide markers which have been reported previously for chicken, pork and horse meat by the use of liquid chromatography-mass spectrometry. Unique peptides specific to both species and muscle protein are now identified, and our results can help solving problems with cases of counterfeiting of meat products across the globe. The research may be relevant to all stakeholders of the food chain: consumers, food industry, legislators and regulatory authorities. Direct ambient surface MS offers a valuable alternative to the existing methods, such as ELISA tests and more time-consuming PCR tests and LC-MS based methods. With the rapid advance of high resolution/accurate mass spectrometry, these ambient techniques may open up new possibilities for rapid analysis of less abundant proteins and be tangible contribution to reduction of food fraud/contamination problems that have arisen in Europe (and elsewhere) in recent years.Contact details:1. Professor David Barrett, Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom. E-mail: david.barrett@nottingham.ac.uk. Tel.: +44(0)1159515062. Fax: +44(0)1159515102.2. Dr Magdalena Montowska, Institute of Meat Technology, Poznan University of Life Sciences, Wojska Polskiego 31, Poznan 60-624, Poland. E-mail: magdalena.montowska@gmail.comTel.: +48618487251. Fax: +48618487254 Related documents final1-attach-1-final-workflow.pdf final1-attach-2-final-mva-results.pdf
