RESUMO
Metal speciation analysis in MLPs was carried out in two snails, Marisa cornuarietis and Pomacea bridgesi, in order to investigate them as possible sentinel organisms of heavy metal contamination. To carry out this study snails born in a non-contaminated environment were divided into two groups: a control group and a contaminated one with cadmium administered for 40 days. Subsequently, we investigated the speciation of the induced MLPs in exposed animals in relation to controls. In order to obtain the MLP fraction, cytosols from both snail species where subjected to size-exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. MLP fraction was then separated by anion-exchange (AE)-FPLC using optimal chromatographic conditions for the separation of the different MLP isoforms in both snail species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with ICP-MS. The determination of the amount of metal bound to MLPs was carried out by post-column isotope dilution analysis ICP-MS, finding that the snail M. cornuarietis accumulated higher concentrations of cadmium than P. bridgesi. Thus this first snail could therefore be a better candidate sentinel organism of pollution in natural waters. Identification and characterization of the isoforms separated in M. cornuarietis was carried out for the entire or intact isoforms by MALDI-TOF and then conventional triptic digestion was also carried out to identify the nature of the formed peptides. The presence identification of a MLP isoform of relatively low molecular weight in M. cornuarietis is reported.
Assuntos
Cromatografia por Troca Iônica/métodos , Água Doce/química , Metalotioneína/análise , Metais Pesados/análise , Proteômica/métodos , Caramujos/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Poluentes Químicos da Água/análise , Sequência de Aminoácidos , Animais , Aquicultura , Ecossistema , CoelhosRESUMO
Quantitative proteomics and absolute determination of proteins are topics of fast growing interest, since only the quantity of proteins or changes in their abundance reflect the status and extent of changes of a given biological system. Quantification of the desired proteins has been carried out by molecule specific MS techniques, but relative quantifications are commonplace so far even resorting to stable isotope labelling techniques such as ICAT and SILAC. In the last decade the idea of using element-selective mass spectrometric detection (e.g. ICP-MS instruments) to achieve absolute quantification has been realised and ICP-MS stands now as a new tool in the field of quantitative proteomics. In this review the emerging role of ICP-MS in protein and proteomic analysis is highlighted. The potential of ICP-MS methods and strategies for screening multiple heteroatoms (e.g. S, P, Se, metals) in proteins and their mixtures and extraordinary capabilities to tackle the problem of absolute protein quantifications, via heteroatom determinations, are discussed and illustrated. New avenues are also open derived from the use of ICP-MS for precise isotope abundance measurements in polyisotopic heteroatoms. The "heteroatom (isotope)-tagged proteomics" concept is focused on the use of naturally present element tags and also extended to any protein by resorting to bioconjugation reactions (i.e. labelling sought proteins and peptides with ICP-MS detectable heteroatoms). A major point of this review is displaying the possibilities of using a "hard" ion source, the ICP, to complement well-established "soft" ion sources for mass spectrometry to tackle present proteomic analysis.