Ignacio Ortea

Mass Spectrometry Characterization of Species-Specific Peptides from Arginine Kinase for the Identification of Commercially Relevant Shrimp Species

The identification of commercial shrimp species is a relevant issue to ensure correct labeling, maintain consumer confidence and enhance the knowledge of the captured species, benefiting both, fisheries and manufacturers. A proteomic approach, based on 2DE, tryptic in-gel digestion, MALDI-TOF MS, and ESI-MS/MS analyses, is proposed for the identification of shrimp species with commercial interest. MALDI-TOF peptide mass fingerprint from arginine kinase tryptic digests were used for the identification of seven commercial, closely related species of Decapoda shrimps. Further identification and characterization of these peptides was performed by CID on an ESI-IT instrument, database search and de novo sequence interpretation, paying special attention to differential, species-specific peptides. Fisheries and manufacturers may take advantage of this methodology as a tool for a rapid and effective seafood product identification and authentication, providing and guaranteeing the quality and safety of the foodstuffs to consumers.

Selected tandem mass spectrometry ion monitoring for the fast identification of seafood species

Selected tandem mass spectrometry (MS/MS) ion monitoring (SMIM) is the most suitable scanning mode to detect known peptides in complex samples when an ion-trap mass spectrometer is the instrument used for the analysis. In this mode, the MS detector is programmed to perform continuous MS/MS scans on one or more selected precursors, either during a selected time interval, or along the whole chromatographic run. MS/MS spectra are recorded, so virtual multiple reaction monitoring chromatogram traces for the different fragment ions can be plotted. In this work, a shotgun proteomics approach was applied to the detection of previously characterized species-specific peptides from different seafood species. The proposed methodology makes use of high intensity focused ultrasound-assisted trypsin digestion for ultra fast sample preparation, peptide separation and identification by reverse phase capillary LC coupled to an ion-trap working in the SMIM scanning mode. This methodology was applied to the differential classification of seven commercial, closely related, species of Decapoda shrimps proving to be an excellent tool for seafood product authentication, which may be used by fisheries and manufacturers to provide a fast and effective identification of the specimens, guaranteeing the quality and safety of foodstuffs to consumers.

Arginine kinase peptide mass fingerprinting as a proteomic approach for species identification and taxonomic analysis of commercially relevant shrimp species.

A proteomic approach aimed at species identification and taxonomic analysis of shrimp species of commercial interest is presented. Six different species belonging to the order Decapoda were considered. Preliminary, two-dimensional gel electrophoresis (2-DE) analysis of the sarcoplasmic proteome revealed interspecific variability in the isoelectric point (pI) of arginine kinase. For this reason, arginine kinase spot was selected as a potential molecular marker and subjected to tryptic digestion followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting (PMF) analysis. Arginine kinase PMF allowed the differentiation of the six species studied. Four samples of commercial origin obtained in local markets were analyzed to validate the methodology. The PMF cluster analysis also provided information about the phylogenetic relationships in these species. The application of this methodology may be of interest for the differentiation and taxonomic analysis of shrimp species complementing DNA-based phylogenetic studies.

Investigation of production method, geographical origin and species authentication in commercially relevant shrimps using stable isotope ratio and/or multi-element analyses combined with chemometrics: An exploratory analysis

Three factors defining the traceability of a food product are production method (wild or farmed), geographical origin and biological species, which have to be checked and guaranteed, not only in order to avoid mislabelling and commercial fraud, but also to address food safety issues and to comply with legal regulations. The aim of this study was to determine whether these three factors could be differentiated in shrimps using stable isotope ratio analysis of carbon and nitrogen and/or multi-element composition. Different multivariate statistics methods were applied to different data subsets in order to evaluate their performance in terms of classification or predictive ability. Although the success rates varied depending on the dataset used, the combination of both techniques allowed the correct classification of 100% of the samples according to their actual origin and method of production, and 93.5% according to biological species. Even though further studies including a larger number of samples in each group are needed in order to validate these findings, we can conclude that these methodologies should be considered for studies regarding seafood product authenticity.

Food authentication of commercially‐relevant shrimp and prawn species: From classical methods to Foodomics

Although seafood species identification has traditionally relied on morphological analysis, sometimes this is difficult to apply for the differentiation among penaeid shrimps owing to their phenotypic similarities and to the frequent removal of external carapace during processing. The objective of this review is to provide an updated and extensive overview on the molecular methods for shrimp and prawn species authentication, in which several omics approaches based on protein and DNA analysis are described. DNA‐based methods include the amplification by PCR of different genes, commonly the mitochondrial 16S ribosomal RNA and cytochrome oxidase I genes. A recently described method based on RFLP coupled to PCR turned out to be particularly interesting for species differentiation and origin identification. Protein analysis methods for the characterization and detection of species‐specific peptides are also summarized, emphasizing some novel proteomics‐based approaches, such as phyloproteomics, peptide fragmentation, and species‐specific peptide detection by HPLC coupled to multiple reaction monitoring (MRM) MS, the latter representing the fastest method described to date for species authentication in food.

Species identification of the Northern shrimp (Pandalus borealis) by polymerase chain reaction-restriction fragment length polymorphism and proteomic analysis

Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was designed based on the amplification of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-ion trap (ESI-IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results.

Closely Related Shrimp Species Identification by MALDI-ToF Mass Spectrometry

Tiger prawn, Penaeus monodon, and banana white prawn, Fenneropenaeus indicus—two of the most important commercial shrimp species—share world distribution, habitat, and similar morphometric characters. Because of commercial regulations about labeling and traceability requirements, species authentication methods are of great interest to fisheries and seafood industries. In this study, peptide mass fingerprints of both species are compared, using Matrix Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) after trypsin digestion of a protein resolved by two-dimensional gel electrophoresis. Though low variability among these species is observed, two peptides of mass 1077.6 and 1205.7 Da are found to be useful to differentiate them.

Food Authentication of Seafood Species

Abstract Food components are regularly adulterated, either deliberately or unintentionally, and this may lead to substitutions of a species by a similar but lower-quality counterpart, mislabeling of products, and therefore to commercial fraud. The authentication of food products is a request from consumers and industry, and accordingly global food regulations have been implemented in order to fight against food adulteration and mislabeling. Many different instrumental techniques have been proposed for species authentication in foodstuffs as tools to comply with food labeling regulations and policies. Although traditional methods such as liquid and gas chromatography, nuclear magnetic resonance, infrared and fluorescence spectroscopy, capillary electrophoresis, and more recently, immunological technologies (e.g., ELISA) and DNA approaches are still being used, they are now being complemented by proteomics-based approaches employing electrophoretic and mass spectrometry methodologies. In this chapter, we offer a comprehensive and updated overview of the proteomics-related studies that have been applied to date for the species authentication in seafood products.

A Method to Compare MALDI--TOF MS PMF Spectra and Its Application in Phyloproteomics

The suitability of a proteomic approach was explored to establish phylogenetic relationships among closely-related species. Decapoda penaeid shrimps were chosen as case study because these species have been widely studied and their phylogenetic relationships have been inferred by extensively validated methods, among which mitochondrial DNA-based PCR studies have provided relevant information Thus, MALDI-TOF mass spectrometry (MS) peptide mass fingerprinting (PMF) of arginine kinase was performed, this enzyme being selected from the sarcoplasmic proteome of such species due to the interspecific variability of their pI values. The presence or absence of selected peptides in the MS spectra was used as a molecular marker for phylogenetic analysis. Based on the cluster analysis of the MALDI-TOF PMF spectra obtained, a dendrogram was generated which could be validated with those obtained using DNA-based methods.