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Development of a proteomics method for meat speciation in heavily processed foodstuffs - FA0138

Description
This bid aims to fill a large capability gap in relation to the detection of undeclared meat in highly processed products such as corned beef. This gap was highlighted during the recent horsemeat crisis. Whilst DNA based detection technologies are in place to test for horse meat in products made with relatively unprocessed meat (e.g. microwave lasagna), most canned foods such as corned beef, cannot be reliably tested for the presence of horse meat using DNA testing methods due to the negative impact on these processes on DNA integrity. Therefore alternative approaches are needed. Proteins possess very diverse physico-chemical properties compared to DNA and some of them will survive extreme heating and pressure conditions. We therefore propose a proteomics approach whereby state-of the art methodology will be used to identify proteins that persist under processing conditions where DNA is largely destroyed (e.g. canning). The technologies developed as the outputs to this proof of concept project will be applicable for use by enforcement authorities and following a successful conclusion, further phases of work will result in routine testing procedures that can be employed by local authorities to test not just for horse meat, but for a range of animal species in processed foods.

This project will use proteomics methodology to identify proteins or peptides that survive heavy processing and that contain stretches of amino acid sequences that are unique to different species of meat producing animals and therefore suitable for meat speciation. As a proof of concept, the focus of the work will be the identification of specific peptides as markers of horse meat in heavily processed foods. An initial discovery phase using a processed meat product (e.g., corned beef) will be carried out where proteins will be extracted and separated by gel electrophoresis. A few proteins clearly resolved in the gel will be excised, digested into peptides and analysed by mass spectrometry technology in order to identify them.

Once we have identified a selection of proteins that survive the processing, bioinformatics will be used to compare their amino acid sequences across a range of relevant meat animal species, including horse, to identify stretches of the proteins where species specific sequences are found (unique marker peptides). Samples of processed horse meat will then be extracted and digested to be analysed by liquid chromatography- tandem mass spectrometry (LC-MS/MS) in order to confirm the sequences of the predicted markers peptides.

Although we will focus on horse peptides for this initial work, the approach proposed here is non-targeted, i.e. it does not use previous knowledge of the proteins to be detected. A non-targeted approach has the advantage of being able to identify new proteins and peptides that persist in these types of foods, unveiling new peptide markers not just for horse meat, but also for other species. It is anticipated that peptides derived from different proteins will be identified, which will offer the opportunity for future development of robust speciation methods based on simultaneous detection of several proteins.
Objective
To identify proteins present in a heavily processed food product (Duration: 3 months)
Corned beef (or another heavily processed meat product selected in agreement with Defra) will be homogenised, de-lipidised and total protein will be extracted. Proteins will be resolved by electrophoresis and any protein bands/spots that can be clearly detected will be excised from the gel and subjected to enzymatic digestion followed by MALDI-TOF MS analysis for protein identification - Sample preparation: Fera; Gel and MALDI-TOF: UoY.
2. To perform BLAST analysis of the amino acid sequences of the proteins identified in objective 1 and select horse specific peptides - Fera, (Duration: 1 month).
The amino acid sequences of the proteins identified will be compared with orthologous sequences from other relevant animal species in order to identify polymorphic peptide sequences that are unique to horse. This will also unveil unique sequences for other species that may be of interest.
3. To evaluate the detection of the selected peptides by LC-MS/MS (Fera, 3 months)
The identification of specific marker peptides in the previous objectives is based on MS data (mass information) and on amino acid information from protein databases. During this objective MS/MS data will be generated in order to confirm peptide sequences. Whole protein extracts from processed horse meat will be digested and analysed by liquid chromatography-MS/MS to obtain sequence information of the unique peptide masses identified in the previous objectives. These results will also indicate which marker peptides are readily detectable after protein extraction without the need for previous protein electrophoretic separation as used in the discovery phase (Objectives 1 and 3). Peptides that can be directly detected in this way will be the best markers for routine speciation tests.
4. To validate the horse meat marker peptides in highly processed meat products spiked with horse meat - Fera, (Duration: 9 months).
This objective is optional. It will be carried out if sufficient funds are available to validate the horse (and other meat species) marker peptides identified in Objectives 1-4 when horse meat is present in other meat products.
Project Documents
• EVID4 - Final project report : FA0138 Evid4   (14050k)
Time-Scale and Cost
From: 2013

To: 2014

Cost: £30,799
Contractor / Funded Organisations
F E R A (FERA)
Keywords