Metalloproteomics studies

Metalloproteomics is a new subject focusing on the distributions and compositions of all metalloproteins in a proteome (metalloproteome), their structural and functional characterization and their structural metal binding moieties. The specificity of metalloproteomics studies demands the need for a description of the metal-binding sites, metal stoichiometry, and metal-depen-dent structure or conformation changes as well as the identifieation and quantification of the metalloproteins. The metalloproteome can be considered as not only a subset of the metallome, but also a very important subset of the proteome. 

Figure 1.7 Roadmap for metalloproteomics studies used in the Laboratory of Nuclear Analytical Techniques of Institute of High Energy Physics, China. 2007 The Royal Society of Chemistry.

X-ray absorption spectroscopy (XAS), especially extended X-ray absorption fine structure (EXAFS), may provide an alternative tool for determining the local structure around certain atoms at a resolution of 10 to 10 nm without the requirement for crystalline samples. For example, Hg in human hair and blood samples from long-term mercury-exposed populations has been studied using EXAFS and structural information such as bond distances and coordination numbers of Hg were obtained. Further, EXAFS can provide a refinement of the structure determined from X-ray crystallography since EXAFS has higher spatial resolution than X-ray crystallography especially in local structures. More detailed information about XAS and its application in metallomics and metalloproteomics study can be found in Chapter 6 (X-ray Absorption Spectroscopy) in this book. 

Table 1.5 Features of the main nuclear analytical techniques for chemical element imaging, quantification, and speciation in metallmoics and metalloproteomics studies. 

X-ray fluorescence is a non-destructive and multielemental analytical technique. Because of its excellent analytical sensitivity and spatial resolution under micro-beam conditions, the technique is capable of microscopic analysis, supplying information about two-dimensional (2D) distributions of trace elements. The technique can, thus, be used for imaging trace elements in biological specimens, and for the direct determination of trace elements in protein bands after slab-gel electrophoresis (GE), which is the benchmark for high-resolution protein separation, particularly in 2D format. Therefore, XRF is a useful technique for metallomics and metalloproteomics studies. 

In comparison with other ionization sources, ESI represents an even softer ionization technique and causes no fragmentation of analyte ions. A major benefit of the generation of multiply charged ions from polypeptides is that they can be readily analyzed with a less sophisticated instrument with limited mass range, such as a quadrupole. A serious problem is its poor tolerance to matrix, such as mobile-phase buffers, when ESI-MS is used for metallomics and metalloproteomics studies. Since the MS response significantly depends on solvent and sample composition, ion signal intensities of a given analyte do not necessarily correlate with its concentration in samples. Therefore, the internal standards are essential for the quantitative analysis. Another solution is the use of ICP-MS as complement.The detailed methods will be described later. 

At present, isotope techniques combined with ICP-MS and ESI-MS have been successfully applied for metallomics and metalloproteomics studies, because of... 

In general, the fingerprints techniques by XANES are widely used in metal-lomics and metalloproteomics studies. More studies are exemplified as Se metabolism of the purple bacterium Rhodobacter sphaeroides, reduction of As in Indian mustard, Fe oxidation states in tissues,Mn in mitochondria isolated from brain, liver, and heart,Cu and Zn intake by Bradybaena similaris (land snail), Ni spedation in hyperaccumulator and non-accumulator Thlaspi... 

In the metallomics and metalloproteomics studies, spatial analysis of absorbing atom of interest is also fascinating for elucidating the uptake and bio-transformation of different species of elements. An example studied the transformation of arsenic (Na2HAs04 or NaAs02) by the arsenic hyperaccumulator, Cretan brake Pteris cretica L. var nervosa Thunb). It was found that As tended to be reduced to As after it was taken up into the root, and arsenic was kept as As when it was transported to the above-ground tissues like petioles and pinnas. However, this example shows the spatial analysis at bulk scale therefore, this kind of XAS techniques can be called bulk-XAS. Using a microscopic mode of XAS, it is possible to obtain the information provided by bulk XAS at a spatial resolution of only a few micrometers or even... 

Despite the wide application of XAS in metallomics and metalloproteomics studies, both theoretical and experimental improvements are still needed. In... 

It is evident that this is a multidisciplinary research field. The metallomics and metalloproteomics studies will not only be beneficial to professional scientists and graduate students in the above fields, but also be hot in medical fields to find the reason how diseases happen. Thus, besides providing useful... 

In metalloproteomics, there has been progress in recent years in developing theoretical methods to study protein structures locally at metal-binding sites and at locations of catalytic activity. The use of quantum chemical calculations can supplement experimental data of PX and can also be used to interpret the structures, e.g. to decide the protonation state of metal-bound ligands. By providing subatomic resolution information that is local to the metal environment, XAS is a valuable ally of computational chemistry. 

Taken together, nowadays we are able to study iron metabolism and distribution at the tissue, cellular, subcellular, or even molecular level by NATs independently or by combining NATs with a variety of pre-separation procedures. The improvement and upgrade of the qualitative and quantitative analytical techniques are the challenges for further progress on the species study at molecular level and the promotion for the development of metallo-genomics, metalloproteomics, and metallomics of iron. 

Application of Integrated Techniques for Micro- and Nano-imaging Towards the Study of Metallomics and Metalloproteomics in Biological Systems... 

In this chapter, we will briefly outline the basic principles of diflerent imaging methods and then focus on some examples of practical application for the study of metallomics, metalloproteomics and the interaction between metals and organisms. 

For the MSI techniques, they can also be applied in metallomics and metalloproteomics with XRF and PIXE. But compared to these two nondestructive techniques, MSI is a partly complete or complete devastating technique to the sample. That is, when the sample is measured over, we can not use it to do other measurements. The most advantage of XRF and PIXE over MSI is their non-destructive and non-invasive characteristics with little or no damage to the sample. So XRF and PIXE are more suitable to study biological samples than MSI if the samples need other further studies. 

In conclusion, with the development of imaging techniques, high-throughput techniques of 2D or 3D features are required for metallomies metallopro-teomics. The development of nuclear or non-nuclear analytieal imaging teeh-niques will greatly accelerate the study of metallomies and metalloproteomics. 

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