SIGLAC · High throughput analytical screening for metal-protein complexes in the biological environment

Final Activity Report Summary - SIGLAC (High throughput analytical screening for metal-protein complexes in the biological environment)

Trace elements play an important role in biochemical processes. Metabolism of the trace elements, in particular their binding to proteins in a biological system is of great importance in biochemical, toxicological and pharmacological studies. The knowledge of the specific binding mechanisms of metals within the soil, water and plants has significant implications for delineating potential bioavailability and/or release of metals. The identification of metal complexes is important to improve understanding of the tolerance and transport mechanisms in plants, which are for instance essential to phytoremediation of the heavy-metal contaminated soil as well as for risk assessment of contaminated foodstuffs. These are only some of the reasons why interest in the speciation of protein-bound trace elements is growing. Cadmium is a non essential element highly toxic to plants and animals. The concentration of Cd in foods depends on many parameters such as ability of the species to absorb and accumulate Cd, the availability of Cd in the soil, which is itself influenced by soil characteristics (pH, cation exchange capacity). Some leafy crops like lettuce or spinach and root crops such as carrots or parsnip are able to accumulate more than other food plants. Furthermore, non-nutritional species such as Arabidopsis halleri can hyperaccumulate Cd and can be used to clean up the contaminated soil by using the phytoremediation method. However, phytoremediation suffers from a lack of an understanding of the basic physiological, biochemical and molecular mechanisms involved in heavy metal hyperaccumulation. This leads to difficulties with the optimisation of the phytoextraction technique and other commercial applications. Therefore, there is a need for the development of improved analytical methods to provide a deeper insight into heavy metal accumulation. The objective of this work was to develop (i) a suitable sample preparation method for the provision of high yields of intact metal-binding proteins; (ii) an analytical methodology suitable for successful screening of metal containing proteins using laser ablation (LA) as a sample introduction system for off-line coupling of non-denaturing gel electrophoresis to inductively coupled plasma mass spectrometry (ICP-MS). The model system used in this study was Spinacia oleracea L. and Arabidopsis halleri treated with Cd. This element was chosen as an example of a highly toxic metal. We have elaborated a new strategy for in-vivo screening of cadmium and zinc containing proteins which consists of the extraction of proteins by application of an ultrasonic homogeniser and separation of the metal-binding proteins by use of 1D native PAGE. For comparison denaturing PAGE has been investigated, too. Laser ablation was utilised as a sample introduction system for ICP-MS detection of the metals in the proteins after PAGE separation and blotting onto membranes. In total 3 proteins have been separated which contain higher levels of cadmium and another with zinc was detected in Spinacia oleracea L. plant. The laser ablation as a sample introduction system for off-line coupling of 1D non-denaturing gel electrophoresis to ICP-MS is a successful and promising method for direct screening of metal-binding proteins. So far this method is just qualitative however; the quantification of the Cd-bond to proteins using the same method would be desirable. In order to characterise low molecular weight metal-binding fractions, size exclusion chromatography (SEC) with ICP-MS detection was applied. Few Cd and Zn binding species have been detected in Spinacia oleracea L. and Arabidposis halleri plants. Nano electrospray (ESI) with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was used for identification of the main protein binding with Cd in spinach plant.

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