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Main activities are:Trace elements play a vital role in biochemical processes, being essential components of molecular structure and hence involved in fundamental processes such as catalysis, enzyme regulation and gene expression. At elevated concentrations trace elements may induce toxic effects hence there is a need to monitor and assess heavy metal status/speciation in biological systems. Speciation measurement involves the combination of a separation technique (gel electrophoresis and/or liquid chromatography) - to fractionate the species of interest -followed by element specific detection (ICP-MS)/molecular analysis (MALDI-MS, ESI- MS and MS/MS). Using the combined approach it is possible to fully characterise elemental/molecular species distribution in complex biological samples. Current research focuses on zinc speciation in bacterial and clinical samples. Cultures of wild type E. coli grown under controlled conditions including sublethal Zn concentrations were first subjected to ion exchange chromatography as shown in the LC-MS workflow:
Fractions were collected (every 30 seconds) and subsequently separated in the second dimension by reverse phase chromatography or alternatively analysed off-line by ICP-MS. The LC flow was coupled directly to ESI-qQTOF-MS or instead the fractions were collected (every 30 seconds) and analysed off-line by MALDI-TOF/TOF-MS. As a third strategy fractions following the second dimension of separation were digested and resultant peptides separated by means of reverse phase chromatography coupled to ESI-QIT-MS/MS, which enabled protein identification. Overall the methodology (2DLC) afforded efficient separation of intact species with good reproducibility and a high level of automation. Moreover tryptic digestion permitted MS/MS identification of low abundance species following the 3rd dimension of separation. The E. coli proteome consists of 4,288 open reading frames (ORFs), where predicted Zn-binding proteins account for 5-6% of the total proteome. Clearly this level of sample complexity presents a challenge for a 2D LC separatory scheme. Each E. coli sample separated in the first dimension (ion exchange) generated 61 fractions for 2D separation (reverse phase) (UV chromatograms shown below).
The typical chromatogram of a fraction separated in the 2nd dimension was still very complex. However the peaks in the TIC were well separated and an deconvolution of the selected spectrum was possible. Additionally by performing off-line ICP-MS analysis of a collected fraction it was possible to detect (and quantify) elevated levels of Zn confirming the presence of Zn binding proteins.
Protection of the environment continues to present new analytical challenges as government and regulatory authorities require industry to meet ever tighter environmental standards. The determination of toxic elements including heavy metals and radionuclides in the environment (air, land and water) is an area where CAS has considerable expertise. 1. Air Pollution Monitoring via Tree Bark The measurement of current and past levels of air pollution is important for an improved understanding of the impact of human activity on the environment. As a result of industrial emissions potentially harmful elements such as arsenic, chromium, mercury, lead and uranium are present in the atmosphere as fine airborne particles and the material can deposit and accumulate on tree bark through wet and dry deposition. Elemental analysis of outer bark collected from rural and industrial areas can offer the potential to relate the multi-element data to specific anthropogenic sources, e.g. car exhaust, incineration, steel production, cement production, power generation etc. An important example relates to isotopic analysis of U in the vicinities of nuclear installations. A further contribution to air pollution assessment concerns the elemental mapping of metropolitan Sheffield. For this project some 700 samples of bark were collected from precise locations and the multielement data sets based on XRF analyses were used to construct spatial distribution maps for 15 elements. Such data may have significance in epidemiological research.
Laser ablation (LA) in combination with ICP emission and ICP mass spectrometry provides a direct in-situ spatially-resolved measurement capability thus complementing traditional ICP measurements based on pneumatic nebulisation of liquid samples. The LA technique offers great scope in R&D, since depending on laser operating conditions, bulk, feature and distribution (2D/3D) data can be realised. Thus multiple line rastering via fine scale laser beam (um) across regions of sample surface affords a new route for imaging analyte distribution. This approach is finding new applications in materials, environmental and life sciences. 1. Elemental Imaging of pharmaceutical tablets Analytical techniques that permit rapid assessment of the composition and distribution of drugs and matrix components – the excipients – are of great importance in the pharmaceutical industry. In general the manufacturing process aims to achieve a homogeneous drug distribution with uniformity of matrix composition. Given the complexity of formulations due in part to use of many different excipients – binders, coatings, disintegrants, fillers, lubricants, preservatives – all drugs contain, to a greater or lesser extent, a wide range of elemental constituents including trace elements. Hence there is an opportunity to monitor compositional variations/heterogeneities/impurities in tablets using the laser ablation technique. In this study we have interfaced a NdYag laser ablation system operating at 266nm (Newwave Research) with a direct-reading ICP emission spectrometry (Spectro Arcos ) which permits simultaneous measurement of all elements – B, Br, C, Ca, Cl, Cu, Fe, K, I, Mg, Na, P, S, Si, Ti, Zn - of interest in real time during multiple line rastering of tablets.
2. Imaging of MRI contrast agents Imaging and quantification of MRI contrast agents (CA) in thin tissue sections is potentially a very important new application area for LA-ICP-MS. In collaboration with the MRI research group at Hammersmith Hospital (Prof. J. Bell); imaging of Gd157 in sections of tumour (see figure below), from mice dosed with a novel Gd-liposome based CA, have been performed (1). Co-registration of LA-ICP-MS images with MRI data confirmed Gd distribution in vascular and necrotic areas of tumour. Specific CA localisation has also been observed in sections of kidney, dependant on inclusion of a folate-receptor targeting molecule. Targeted formulations were confined to the major calyx and renal pelvis, whereas non-targeted liposomes showed distribution throughout the entire kidney. This study highlights the benefits of LA-ICP-MS, in determining the ultimate fate of administered agents in vivo. This has implications for the pharmacological study of a wide range of therapeutic agents, whose only requirement is to have an appropriate (i.e. non-endogenous) elemental label.
3. Immunoassay-based elemental imaging A further opportunity for elemental imaging in thin biological tissue relates to the use of metallic probes in the immunohistochemical (IHC) experiment. IHC procedures are in widespread use in the clinical/biochemical field but methodology is considered to be semi-quantitative at best. By incorporating an elemental centre (e.g., Au, rare earth) into the detection antibody it is possible to provide a new route for quantitative measurement and mapping of target antigens. The viability of this approach has been demonstrated for imaging of B amyloid peptide and the cancer biomarkers HER2 and MUC1. The accompanying figure depicts the photomicrograph (LHS) of normal human gastric mucosa stained for MUC-1 antigen using the immunogold technique and corresponding Au distribution by LA-ICP-MS (81 line rasters) (single line rasters on RHS).
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| Centre for Analytical Sciences | The University of Sheffield | Chemistry |