Shrimp detectives
Seafood is a major commodity within the food industry but, as reported recently for meats, there are widespread concerns about food authentication. Is that fish the species it is claimed to be? This question has implications not only for traceability, adulteration and labelling, but also for food safety.
Shrimp are one of the highly commercial seafoods that are harvested freely from the oceans but are also farmed in aquaculture businesses. Unfortunately, they are also one family in which many different species look alike and visual inspection may not be sufficient to differentiate between them. This is particularly the case during processing when the discriminating features are broken off.
The molecular methods that have been devised to allow species identification are centred around mitochondrial DNA analysis and protein analysis and tend to be long-winded. Within proteomics, one group of Spanish researchers has been investigating the use of mass spectrometry, which has the advantage of rapid analysis and the potential for high throughput.
Ignacio Ortea and Jose Gallardo from the Marine Research Institute, Vigo, and Benito Canas from the Complutense University of Madrid, focused on the enzyme arginine kinase, which has been put forward as a potential marker of decapods, the group of crustaceans that includes lobsters, crayfish, crabs and shrimp. Inter-species variations in the amino acid sequences of the enzyme appear to be sufficiently specific for differentiation of the species.
In 2009, the research team published data characterising the arginine kinases from seven closely related species of commercially important shrimp using tandem mass spectrometry of the tryptic digests. Now, they have used those results as the basis of a shotgun proteomics method to permit rapid species identification.
Selected MS/MS ion monitoring
The seven shrimp species were Pleoticus muelleri (Argentine red shrimp), Pandalus borealis (Northern shrimp), Penaeus monodon (giant tiger prawn), Litopenaeus vannamei (Pacific white shrimp), Fenneropenaeus merguiensis (Banana prawn), Fenneropenaeus indicus (Indian white prawn) and Farfantepenaeus notialis (Southern pink shrimp).
Keeping in mind the preference for a fast analytical procedure, the sarcoplasmic proteins extracted from each species were subjected to digestion with trypsin under high-intensity focused ultrasound, which was sufficiently complete within 60 seconds. The resultant peptides were analysed by LC-tandem-MS on an ion trap mass spectrometer.
As the peptides eluted from the HPLC column in a 65-minute gradient, they were scanned by selected MS/MS ion monitoring (SMIM), one of the best methods for detecting and quantifying peptides that have already been sequenced. Continuous MS/MS scans were carried out on seven peptides during the whole HPLC run, one peptide from the arginine kinase of each shrimp species.
The peptides were chosen because they were species-specific. In each case, a unique transition from the doubly charged precursor ion to the product ion was observed and chromatograms were plotted of the selected product ions as a function of retention time.
These provided unambiguous discrimination between all seven species, even those within the same family. One of the main aims, to provide a fast procedure, was also fulfilled. The total time from the arrival of the sample to species identification took less than 90 minutes.
The researchers also studied two commercial samples, labelled as Penaeus species and frozen vannamei shrimp tails, respectively. The labelling appeared to be correct, as the samples were identified by SMIM as Penaeus monodon and Litopenaeus vannamei.
The seven species could also be identified from an offline SMIM analysis, with no initial LC separation. In this case, the same seven selected precursor ions were subjected to MS/MS, even if they were not clearly visible in the full scan spectrum. Then the spectra were visually examined for peaks matching known fragment ions of each peptide. The results agreed with those from the online MS/MS experiments.
The offline method would not normally be recommended due to the need to resolve the complex peptide mixture before mass spectrometric analysis. However, in this case, arginine kinase is highly abundant in the extracts, so its tryptic peptides were also abundant. In addition, the particular mode of shrimp extraction, using water and no salts, markedly reduced the sample complexity.
The SMIM procedure is the fastest published method to date for the unambiguous identification of individual shrimp species, whether in online or offline mode, although the offline method is less amenable to automation.
They will be useful for food producers to fulfil labelling guidelines and to guarantee the safety and quality of their products to the consumer.
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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