Is it LAMB? Meat consumption is one of the highest of all food types across the globe, and food producers are sometimes looking to find more economic and cost effective ways to produce assorted meat products for human consumption. In 2013, news was made when beef was adulterated with horse meat, which has some potentially hazardous side-effects for human consumption (since horses are sometimes treated with medicines that are toxic to humans). Additionally, there was news in Asia of mutton rolls being adulterated with duck and even rat meat, and 'fake lamb' meat was caught being sold in supermarkets (but the product was actually comprised of other meat species). Besides consumer safety, there are ethical and religious concerns related to consumption of some meat products. For example, consumption of pork is of high concern for Islamic and Judaic communities. Labs are now trending to find new approaches to test meat products for authenticity and adulteration to identify exactly what species are present.
Meat speciation in the laboratory has traditionally been performed using either PCR (polymerase chain reaction) or ELISA (enzyme-linked immunosorbent assay). Both techniques are limited in accuracy, sensitivity, and specificity, making the search for alternative analytical approaches to verify meat authenticity very important to food testing laboratories. LC/MS/MS provides an excellent alternative to traditional methodologies to identify and confirm targeted peptide markers associated with different meat species more accurately and reliably than existing methodologies. In this article, we will describe a two-pronged approach using LC/MS/MS to first identify the unique protein markers specific to a meat species using the TripleTOF® 5600, then utilizing the MIDAS workflow (MRM-initiated detection and sequencing, Figure 1) on the QTRAP system to perform survey scans targeting the MRMs of targeted peptide markers of multiple meat species of interest then triggering the acquisition of an EPI spectrum for added peptide confirmation. This approach has been successfully applied to the detection of horse, duck, chicken, beef, pork, and rat meat contamination in food samples ranging from ground meat to mutton rolls (which should only contain lamb meat) purchased from local supermarkets in China. The approach has also been applied in the speciation of gelatin used in candies and pharmaceutical capsules.
Figure 1: The MIDAS™ workflow utilizes MRM-initiated detection and sequencing.
Figure 2: For the detection of horse peptides, MRM initiated acquisition of MS/MS spectra was used to sequence characteristic proteins for added selectivity and specificity in the analysis. The results show that this LC/MS/MS workflow can be successfully applied to testing food products and accurately identifying what meat species are present in those samples to verify their authenticity and integrity. The result is a more accurate, reliable, and direct approach to determining meat authenticity than traditional analytical testing methodologies.
Duck, chicken, lamb, beef, pork, horse, and rabbit meat tissue were purchased commercially. Rat tissue was harvested from rat leg muscle that had been used in previous toxicological experiments. Food samples such as beef, lamb, mutton rolls, gummy bears, and others were purchased from local supermarkets (Europe and China).
Details of the sample preparation for different samples types can be reviewed in the assorted reference publications and application notes1-4, which can vary slightly depending on the matrix. In short, homogenized food samples are extracted with a buffered extraction solution then undergo a tryptic digest. In some cases, the digested extracts are purified using solidphase extraction prior to analysis by LC/MS/MS.
Identifying peptide targets for each species
Initial identification of species-specific peptides from the tryptic digests was done using a shotgun proteomics approach on the TripleTOF® 5600 system coupled to an Eksigent nanoLC or ultraLC system. In these survey scans, an information dependent acquisition (IDA) method is used to trigger the acquisition of TOF-MS/MS spectra associated with all peaks detected in the TOF-MS survey scan. For the gelatin speciation experiments, principal component analysis using MarkerView™ Software was performed to identify the specific markers for each tissue type4. For the experiments testing mutton rolls for authenticity, ProteinPilot Software was used to identify all characteristic proteins and peptides for each meat species. We targeted the highest abundance proteins for each species to ensure best sensitivity and accuracy. Detailed comparisons of proteins and peptides for each species were made, especially since some species have close genetic relationships (such as chickens and ducks or sheep and cows) and have proteins and peptides that differ by only a few amino acids. Final results were confirmed through a blast on the NCBI website to ensure that each identified peptide was totally unique to that meat species to ensure highest specificity for the analysis.
Analysis of meat peptide MRMs using the MIDAS workflow
Once the peptide fragments for each meat species were identified, the MIDAS workflow (Figure 1) was used to create MRM detection methods for analysis using the QTRAP® system. Detailed method conditions can be found in the reference documents below, highlighting the methods for the analysis of horsemeat contamination in beef1, 2, meat species analysis in commercial mutton rolls3, and gelatin speciation in gummy bear candies4. Using QTRAP technology, the mass spec was set-up for electrospray ionization (ESI) utilizing an MRM-triggered EPI method, enabling us to collect full MS/MS spectra associated with each MRM for the highest selectivity in sequence identification. Figure 2 shows the MS/MS spectra collected for horse peptides, which allow for sequencing of the peptides for added specificity and selectivity in the analysis.
Figure 3: The NCBI database is incomplete for duck proteins, resulting in duck samples being identified as chicken in the ProteinPilot search. The MIDAS workflow allows these 2 species to be distinguished (Figure 4).
Figure 4: The LC/MS/MS MIDAS workflow enables us to identify peptide markers for each meat species, and allows us to selectively identify even closely related species. Here we show the analysis for detection of duck peptides, which we detect in the duck sample (top panel) but not in any other meat tissue sample tested, showing the high specificity of the analysis.
Figure 5: Lamb samples purchased from the supermarket were tested for a number of meat species including chicken, duck, and others, to verify their authenticity. Here we show the analysis for duck meat markers in a store-bought sample (top panel), an authentic lamb leg (middle panel), and in an authentic duck tissue sample (bottom panel). Results showed that the store-bought samples were 'fake lamb', and contained approximately 50% duck meat.
Figure 6: The meat speciation approach also works well for gelatin speciation. Here bovine and porcine gelatin markers were analyzed in gummy bears, chocolate candies, and pharmaceutical capsules. While pork gelatin markers were detected in the candies, only bovine markers were detected in the capsules.
A chicken or duck conundrum
The close genetic relationship between chickens and ducks, and the incomplete NCBI database for duck proteins, resulted in database search results in ProteinPilot™ identifying both duck and chicken samples as chicken proteins (Figure 3). From this initial analysis using the database, we were unable to unequivocally identify if the exact fowl species was chicken or duck. However, through the use of the MIDAS™ workflow to selectively identify unique peptide markers for each, we are able to use MRM detection to selectively detect and accurately identify both species (Figure 4). A key benefit to using LC/MS/MS for meat speciation is the selectivity for the mass spec to identify even small differences in amino acid sequences to enable the reliable identification of even closely-related meat species. Speciation results in commercial samples Many commercial samples, from beef, to mutton rolls, to candies, were analyzed using our LC/MS/MS speciation methods to identify what species of meat tissue were present in these samples, and, in some cases, verify how authentic the product was based on its label claims. Figure 5 shows the analysis of a lamb purchased at a local supermarket in China (top panel). The peptides detected in that sample were consistent with the peptide markers for duck meat, suggesting that the samples were falsely labeled as lamb but actually consisted of approximately 50% duck tissue.
Meat authenticity and integrity is a very hot and controversial topic in the news, both for the safety implications but also for the ethical implications associated with consuming falselylabeled food products. LC/MS/MS is emerging as a proven technique to enable labs to speciate meat in assorted food and consumer samples. This article gives a brief overview of how LC/MS/MS can be used in meat speciation, and shows a few key examples of this approach being performed on real samples purchased in local supermarkets.
Get more details on these speciation applications:
Von Bargen, et. al., J. Agric. Food Chem, 2013, 61 (49), 11986.https://pubs.acs.org/doi/abs/10.1021/jf404121b