Characterization and partial sequencing of species-specific sarcoplasmic polypeptides from commercial hake species by mass spectrometry following two-dimensional electrophoresis

Abstract

The Merluccidae family comprises marine species, some of them of high commercial value and others less appreciated, whose commercialization in Europe under the generic name of "hake" is highly remarkable. The potential of proteomics was employed in this study with the aim of achieving the differential characterization of five different hake species: Merluccius merluccius (European hake), M. australis (Southern hake), M. hubbsi (Argentinian hake), M. gayi (Chilean hake), and M. capensis (Cape hake), some of them very closely related. Species-specific polypeptides were observed for the five hake species studied in isoelectric focusing (IEF) and/or two-dimensional electrophoresis (2-DE) high-resolution gels. The peptide mass maps of two polypeptide groups, previously selected by 2-DE analysis as potentially species-specific, were obtained by "in-gel" tryptic digestion, followed by matrix assisted laser desorption/ ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Analysis of group A polypeptides (with pI in the range of 5.0-5.5 and molecular mass of 17 kDa), allowed the differential classification of the hake species into two groups: the East Atlantic coast group and the West Atlantic coast group. Moreover, the peptide mass-maps from the heat-resistant parvalbumin fraction (pI below 4.5; molecular mass <12 kDa) allowed the detection of a peptide characteristic of M. australis not present in the other four hake species tested. A specific 17 kDa protein from M. merluccius was also partially sequenced by nanospray-ion trap-tandem MS, revealing a high homology with rat nucleoside diphosphate kinase A (NDKA). This work opens the way to the application of proteomics to the differential characterization of commercial hake species at the molecular level.

Methods of gadoid fish species identification in food and their economic impact in the Czech Republic: a review

“…Heat resistant sarcoplasmic proteins (parvalbumins) and some myofibrillar proteins (Sotelo et al, 1993;Rehbein et al, 1999;Pineiro et al, 2001) are significant markers for electrophoretic discrimination between heat treated fish species. Several isoforms of parvalbumins -acidic, heat stable, calcium binding and low Mw protein -exist.…”

“…M. merluccius shared one parvalbumin with M. australis and M. hubbsi, and two parvalbumins with M. capensis (Pineiro et al, 1998). Pineiro et al (2001) concentrated on the authentication of five species of hakes M. merluccius, M. australis, M. hubbsi, M. gayi and M. capensis based on differentiation between species-specific sarcoplasmic polypeptides using the 2DE method and subsequent mass spectrometry (MS) of the isolated proteins. Two groups of polypeptides separated by 2DE have been selected (group of polypeptides with pI 5.0-5.5; Mw 17 kDa and parvalbumin fraction of proteins with pI < 4.5; Mw < 12 kDa) for MS analysis.…”

“…Accordingly, further research will be focused on the determination of amino acid composition of the hake homologue of NDKA as a potential compound for identification of fish species of family Merlucciidae (Pineiro et al, 2001). Protein solubility, prevention of their degradation and modification by endogenous proteolytic activity, and removal of interfering substances (if necessary) are necessary prerequisites that basically affect the quality of 2DE results.…”

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“…Protein electrophoresis is a technique used for the study of species genetic structure and the determination of phylogenetic relationships (Focant, Jacob, &Huriaux, 1981 andPineiro, Vazquez, Marina, Barros Velazquez, &Gallardo, 2001). Soluble proteins of muscle sarcoplasm are among the easiest to extract and highly a rich reservoir of species specific and biochemical genetic markers (Tsuyuki et al, 1965, O'Rourke, 1974Ryman, Utter, & Laikre, 1995;Ryman & Utter 1987;Buth and Murphy 1999).…”

Application of DNA-based Techniques for Intraspecies Differentiation of Hake Fish

“…two-dimensional electrophoresis (2DE) was used for the detection of Merluccius merluccius, M. australis, M. hubbsi and M. capensis (Pinerio et al 1998;Pineiro et al 2001). For the identification of Merluccius merluccius urea-IEF , sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) Pineiro et al 1999) and chromatography (Pineiro et al 1997) were employed.…”