José Luis López

Application of proteomics for fast identification of species-specific peptides from marine species.

In this work, a novel approach based on proteomics is applied for the analysis of the three European marine mussel species: Mytilus edulis (ME), Mytilus galloprovincialis (MG) and Mytilus trossulus (MT), which are of interest in biotechnology and food industry. The proteomes of these species are poorly described in databases, are difficult to diagnose, and have a controversial taxonomy, To characterise species‐specific peptides, we compared 51 matrix‐assisted laser desorption/ioization‐time of flight peptide mass maps generated from 6 random selected prominent spots derived from the two‐dimensional electrophoresis analysis of foot protein extracts from several individuals. Minor species‐specific differences in the peptide maps were detected in only one of the spots, corresponding to tropomyosin. Two peptides were unique to ME and MG individuals, whereas another peptide was present only in MT individuals. The sequence of these peptides was characterised by, nanoelectrospray ionization‐ion trap (nanoESI‐IT) tandem mass spectrometry (MS/MS) analysis followed by database searching and de novo sequence interpretation. We detected a single T to D amino acid substitution in MT tropomyosin. Unambiguous and highly‐specific species identification was then demonstrated by analysing peptide extracts from tropomyosin spots by micro high‐performande liquid chromatography (microHPL) ESI‐IT mass spectrometry using the selected ion monitoring configuration, focused on these peptides, in continuous MS/MS operation. Our results suggest that proteomics may be successfully applied for the identification of species whose proteome is not present in databases.

Growth, mortality, pathological conditions and protein expression of Mytilus edulis and M. galloprovincialis crosses cultured in the Rı́a de Arousa (NW of Spain)

Abstract In this article, the growth, mortality, pathological conditions and protein expression of hatchery obtained mussels from pure and hybrid crosses between individuals from three genetically divergent European populations were evaluated in the Ria de Arousa (NW Spain) under raft-suspended cultivation conditions. Progenitors for the crosses were obtained from a Mytilus edulis population from The Netherlands and from two M. galloprovincialis populations located on each of the two sides of a major genetic break, associated with the Almeria–Oran oceanographic front in the Iberian Peninsula. Results indicated that mussels from crosses between individuals of M. galloprovincialis populations have a significantly higher biomass production than those from hybrid crosses between individuals of M. galloprovincialis and M. edulis populations. This different performance was not due to differences in growth rate but rather to the considerably higher mortality, during the warmer season, of the mussels from hybrid crosses. This strong viability selection operating against hybrid mussels with respect to pure M. galloprovincialis crosses under raft cultivation conditions in the Ria de Arousa was very similar to that operating in natural populations of the M. edulis / M. galloprovincialis hybrid zone in SW England, reported in previous studies. Our results also show that the lower viability of hybrid mussels is clearly associated with both higher parasitization by the protistan Marteilia refringens and lower levels of the stress proteins calreticulin and heat shock protein 70. Among the mussels from the different M. galloprovincialis crosses, those from crosses between males and females of autochthonous (Galician) origin are the ones which show a better performance.

Proteomic approach to probe for larval proteins of the mussel Mytilus galloprovincialis.

A proteomic approach was used to search for larval proteins specific to the mussel Mytilus galloprovincialis from Galicia in northwest Spain. The study included both a comparative analysis, through two-dimensional electrophoresis, of protein expression maps of the larvae of the mussel and of 5 abundant and commercially important bivalve species from the region (Ostrea edulis, Cerastoderma edule, Pecten maximus, Tapes decussatus, and Venarupis pullastra) and subsequent mass spectrometric analysis of some of the protein spots. A total of 18 spots were selected and isolated from gels of M. galloprovincialis larvae. From their relative position on the electrophoresis gels, 6 of these were clearly exclusive to the mussel species. However, it was not clear whether the other spots were shared by other species. To overcome this ambiguity, first an analysis using matrix assisted laser desorption ionization with time-of-flight (MALDI-TOF) was conducted on the 6 spots of Mytilus that could possibly be shared with only one species. The peptide mass fingerprinting was completely different for the proteins compared. This result confirmed that the 6 proteins were exclusively mussel proteins, but demonstrated the utility of this approach when working with species that are poorly represented at the protein level in databases.

A better understanding of molecular mechanisms underlying human disease

This review summarises and discusses the degree to which proteomics is contributing to medical care, providing examples and signspots for future directions. Why do genomic approaches provide a limited view of gene expression? Because of the multifactorial nature of many diseases, proteomics enables us to understand the molecular basis of disease, not only at the organism, whole‐cell or tissue levels, but also in subcellular structures, protein complexes and biological fluids. The application of proteomics in medicine is expected to have a major impact by providing an integrated view of individual disease processes. This review describes several proteomic platforms and examines the role of proteomics as a tool for clinical biomarker discovery, the identification of prognostic and earlier diagnostic markers, their use in monitoring the effects of drug treatments and eventually find more efficient and safer therapeutics for a wide range of pathologies.

Proteomics and multilocus sequence analysis confirm intraspecific variability of Vibrio tapetis

Vibrio tapetis is the etiological agent of brown ring disease (BRD) in clams. Phenotypic, antigenic and genetic variability have been demonstrated, with three groups being established associated with host origin. In this work we analyze the variability of representative strains of these three groups, CECT 4600(T) and GR0202RD, isolated from Manila clam and carpet-shell clam, respectively, and HH6087, isolated from halibut, on the basis of the whole proteome analysis by 2D-PAGE and multilocus sequence analysis (MLSA). A quantitative analysis of the proteome match coefficient showed a similarity of 79% between the clam isolates, whereas fish isolate showed similarities lower than 70%. A preliminary mass spectrometry (MS) assay allowed the identification of 27 proteins including 50S ribosomal protein L9, riboflavin synthase β subunit, ribose-phosphate pyrophosphokinase and succinyl-CoA synthase α subunit. The MLSA approach gave similar results, showing a 99.4% similarity of the clam isolates, which was higher than that observed between the fish isolate and either clam strain (98.2%). The topology of the maximum parsimony tree, obtained from 2D-PAGE analysis, and the phylogenetic tree, constructed with the maximum likelihood algorithm from concatenated sequences of 16S rRNA gene and five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD), was very similar, confirming the closer relationship between the two clam isolates.