Differentiation of Four Tuna Species by Two-Dimensional Electrophoresis and Mass Spectrometric Analysis

Species belonging to the genus Thunnus are pelagic predator fishes, commonly known as tuna. The species within this genus are of commercial value, and six of them are considered the most valued in world trade (D.M., MIPAAF, 31 Gennaio 2008). Thunnus species originate from a variety of geographic areas, and for this reason the different species can be characterized by the presence of different biological contaminants and sensory characteristics. The species Thunnus thynnus has a higher quality and commercial value due to its excellent organoleptic features. Tuna species are usually consumed as fillets or processed products. The loss of the external anatomical and morphological features makes the authentication of a fish species difficult or impossible and enables fraudulent substitutions (Marko et al., 2004). Species substitution is very common in fish products, due to the profits resulting from the use of less expensive species. For species of tuna, substitutions have both commercial and health implications (Agusa et al., 2005; Besada et al., 2006; Storelli et al., 2010), thus, analytical techniques to differentiate fish species are essential. The development of suitable analytical methods for fish species identification in prepared and transformed fish products is of great interest to enforcement agencies involved with labelling regulations and the authentication of fish in various products to prevent the substitution of fish species (Mackie et al., 2000; Meyer et al., 1995). Several biochemical techniques enable the study and identification of fillet or minced fish species. Among these methods, isoelectric focusing (IEF) (Etienne et al., 2000; Rehbein et al., 2000; Renon et al., 2001;), capillary zone electrophoresis (Acuña et al., 2008), and amplification of selected DNA sequences by the polymerase chain reaction (PCR) have been used for the identification of certain groups of fish species (Espiñeira et al., 2008; Hubalkova et al., 2008; Pepe et al., 2005, 2007; Trotta et al., 2005). Presently, PCR is the most frequently used technique, as DNA is heat-stable and resistant to heat treatments that may be applied to the tuna during processing. However, obtaining an accurate species identification is very difficult if the species show a high degree of homology as Thunnus does (Chow & Kishino, 1995; Lopez & Pardo, 2005; Michelini et al., 2007; Pardo