The rapid multiple reaction monitoring (MRM) mass spectrometric method is for detection and relative quantitation of the adulteration of meat with an undeclared species.
They focused on myoglobin and relied solely on derived peptides as the marker peptides for different meat species instead of using a shotgun-type proteomic approach.
It is relatively heat-stable, suggesting that species testing based on myoglobin will work for raw and cooked meat products.
Researchers examined horse in beef, beef in lamb, and pork in lamb due to potential relevance to food fraud.
The team found the method is capable of detecting 1% (w/w) of one meat mixed with another across the meats studied. Specificity is guaranteed by the ability to detect at least four transitions, they added.
Timeline and progress of work
Dr Kate Kemsley said the team started the work in the summer of 2014 after securing internal funding at IFR for one year.
After promising results, they applied for further funds from the EU Food Integrity project which had a call for projects in topical research gaps. They secured funding and started the two year project in March 2016, with partner researchers in Stuttgart and Gdansk.
“We have had great success so far with ready meal lasagnes and beef burgers that were at the heart of the horse meat scandal. The next big challenge, which is also an issue for DNA testing, is canned foods.” she told FoodQualityNews.
“The myoglobin protein is present in a wide range of meats. It is the protein that colours meat red, but there are tiny differences in amino acids between species which are easy to pick up with targeted mass spectrometry.
“Myoglobin is also present in white meat at lower levels. We know we can pick up low levels of adulteration in pork, beef and lamb with horse. In principal, we will be able to do the same test for other things like venison and rabbit.”
Dr Kemsley added when completed the aim is to roll it out as a method and set people up with the instrumentation and training to be able to do it.
A look at the science
Myoglobin in beef differs from horse by 18 amino acids, the building blocks of all proteins. This means that if the beef and horse proteins are broken up in the same way, some of the matching fragments have different masses.
Protein extracted from a meat sample is chemically chopped into fragments, called peptides, using an enzyme. The peptide soup is fed into a mass spectrometer that is tuned to measure the masses of only a handful of selected peptides.
If a burger contains only beef then only beef peptides will appear. But if there is horse then some horse peptides will show up too. The relative hit rate of the horse and beef peptides give an estimate of how much horse has been added. The procedure takes around two hours.
Dr Kemsley said some species – such as horse and donkey – share exactly the same myoglobin, but there are other proteins that could be used to distinguish them.
“The number of species likely to be present in the food chain is not vast. You might struggle with tiny percentage contamination levels such as if you were testing for religious reasons like if pork was halal, or arbitrarily small like if allergens from a nut processing line were enough to carry over, but fraud is not at these contamination levels as it would not make economic sense.”
The myoglobin (Mb) level of an animal varies with its age and with muscle type, according to the study.
“In some ways this is not as serious as it might seem since a realistic average Mb value can be established for commercially supplied meats of known provenance. However, for an adulterant meat the provenance cannot be assumed and the relevant level of Mb is more uncertain,” according to the research.
“As a result, a measurement of relative levels of Mb arising from two species in a sample cannot be mapped with certainty to an exact percent (w/w) mix of meats.
“In summary, the target for ratios of transition peak areas is a ratio of corresponding Mb levels from two species. The ratio of Mb levels then maps to an estimate of percentage (w/w) of meats informed by the documented range of Mb levels of the two species in the mix.”
Mass spectrometry is limited by the scan time but throughput could look like 10-20 a day and it certainly avoids the three day turnaround time for results, said Dr Kemsley.
“Broad basket testing takes a longer time. If you are focusing on routine testing for a specific product you could shave time off the mass spectrometry run. All manufacturers are eyeing routine testing and bringing machines down in complexity and size,” she added.
“At the high-end you can do a lot on a range of issues, at the other end you do the specific analysis you are interested in. You need to have a clear idea of what you want to measure and what question you want to answer.
“Mass spectrometry still needs to become accepted. It is regarded as high-end and expensive and difficult to use, but as it gets cheaper and better adapted for particular tasks we will see an uptake.”
Source: Analytical Chemistry
"Meat Authentication via Multiple Reaction Monitoring Mass Spectrometry of Myoglobin Peptides"
Authors: Andrew D. Watson, Yvonne Gunning, Neil M. Rigby, Mark Philo, and E. Kate Kemsley