SCOTSMAN (SCOttish Trace element Speciation & Metabolomics Analytical Network) - a request for a high resolution ICP-MS to accomplish HPLC-ES-qTOFMS

Lead Research Organisation: University of Aberdeen
Department Name: Chemistry


Medical diagnosis and the resulting treatment will improve the results significantly when a more personalized system for health assessment is implemented. This can be achieved by providing detailed information about the metabolic status of individuals. The use of metabolomic data to predict the health trajectories of individuals will require bioinformatic tools and quantitative reference databases. For example protein phosphorylation is probably the most important regulatory event in eukaryotes. Many enzymes and receptors are switched 'on' or 'off' by phosphorylation and dephosphorylation. Antibodies can be used as powerful tools to detect whether a protein is phosphorylated at any particular site. Such antibodies are called phospho-specific antibodies; hundreds of such antibodies are now available. They are becoming critical reagents both for basic research and for clinical diagnosis. Approaches to identify and more importantly quantify phosphorylated proteins, like mass spectrometry-based proteomics, are becoming increasingly important for the systematic analysis of complex phosphorylation networks. However, most of them lack the ability to identify the phosphorylation status rapidly and accurately. Furthermore, other post translational modification such as sulphurylation and the redox status of translational proteins and selenoproteins could give vital information about the metabolic status of an individual. Two challenges lie ahead for the bioanalytical community; the separation of the complex mixtures of metabolites, peptides and proteins and their quantitative determination. Most methods can only cover one of the challenges. Here, with this proposal, we seek funding to complete the world-wide unique set-up the SCOttish Trace element Speciation & Metabolomics Analytical Network (SCOTSMAN), a new combination of chromatography and/or electrophoresis and dual mass spectrometry to develop a rapid separation technique which is capable of online identification and quantification of metabolites and proteins which have been labelled or tagged in a complex matrix of organic compounds which do not contain an hetero-element. Hence, this method is able to pick out the needles in the haystack. This set-up will be able to quantify biomolecules containing a hetero-element such as phosphorous or sulphur or metals and metalloids such as copper, selenium and arsenic. Using element-specific detection coupled with high resolution mass separation, the requested instrument is capable of quantifying the compounds at ultra-trace level which is relevant for background studies and non diseased individuals. Since the instrument response is not dependant on the compound itself, it can be used to quantify the element in the introduced sample without having the exact compound as a standard. If that analyser is now coupled to a separation method online, the unambiguous quantification of the compound carrying the tag or label can be done directly. When identification of certain metabolites is of importance, the second complementary molecular mass analyser (already in place) will provide accurate data on the mass of the molecule simultaneously. This information is vital to deduce molecular formula. Altogether this proposal, supported by the manufacturer and a charity organisation has an extremely good add on value, since the requested instrument will be coupled directly with additional complementary mass analyser with similar calibre to built this unique analytical set-up for biologists, plant physiologists, microbiologists, researcher interested in systems biology and pharmacologists.

Technical Summary

The technology of high resolution elemental mass spectrometry has matured over the last decade but its potential in life sciences has not been recognized so far. Although quadrupole ICP-MS have been used for decades as detectors for HPLC in order to determine low molecular mass metal species. But it was only when this set-up was combined directly with electrospray MS, that it became a powerful tool for the identification and quantification of biomolecules. However, this set-up has also shown its limitations, since the quadrupole ICPMS are not capable of determining the more common hetero-elements in bioligands such as phosphorous and sulphur. Although the technology is capable of detecting those non-metals with high sensitivity and interference-free by using double focussing sector field ICP-MS, it has never been used in the combination with HPLC and electrospray mass spectrometry. We propose to combine the requested high resolution ICPMS (Element 2) with an ES-qTOFMS for measuring the accurate mass or molecular formula of an eluting compound directly when online coupled to HPLC and to quantify the compound by utilising the independence of plasma responses of different species containing one hetero-element. Here we capitalize on our experience in combining dual mass spectrometry facilities for liquid chromatography in order to identify and quantify unstable intermediates and compounds of low abundance in cell extracts. This set-up makes it possible to identify new biomolecules and quantify known species of importance in a mixture of hundreds of untagged molecules in the extracts. In particular the quantification of post translational modification of proteins by phosphorylation and sulphurylation may become a routine technique.
Description This grant was used to accomplish an analytical facility which was at the end of the grant world-wide unique by acquiring a high-resolution element mass spectrometer (HR-ICP-MS), which was used simultaneously in parallel with a molecular mass spectrometer as detectors for a separation unit. The ICP-MS is used for compounds which contain a trace element and is able to detect these compounds in a mixture of hundreds or thousand biomolecules. Hence, ICP-MS makes it possible to screen for the needle in the haystack or the arsenic in a rice grain. It does not only identify a compound, it can also be used to tell how much of the compound is in a sample (absolute quantification). The molecular mass spectrometer used simultaneously will give the molecular structure of the trace element. This facility can be used for biological extracts or environmental samples. As promised in the application this facility has been used by researchers from different fields as illustrated in the list of published work since 2009: biochemistry (McEwan et al. 2011), plant physiology (Ye et al. 2011), archaeology (Meharg et al. 2012), food and analytical chemistry (Amayo et al. 2011).

One feature of the HR-ICP-MS is that the co-called hetero-elements in biomolecules such as sulphur or phosphorous can be measured in traces without any interferences. Since such elements are in almost all biomolecules, the element can be used as a label or tag. When now the total amount of the element in a compound has been determined and for example the amino acid sequence of a protein is known, a protein can be quantified with a small uncertainty. We used this methodology to determine the stoichiometry of zinc in an integrase by using this methodology (McEwan et al. 2011). On the other hand small a mixture of complex arsenic containing lipids in fishmeal count not only be identified but also be quantified (Amayo et al. 2011). This opens up the opportunity to analytical chemists to validate their methodology by checking mass balances of the element of interest.

Using this combination of mass spectrometers, which are used in tandem makes it possible to identify labile complexes of toxic elements in plants. We have been able to determine these compounds and identified a biomarker for plants which are studied for their low translocation rate from roots to shoots (Liu et al. 2010). This is important to identify crops which tend not to accumulate toxic elements in their edible parts such as rice. Since the high amount or arsenic in rice is of toxicological concern, metrology institutes organised a world-wide proficiency test for arsenic in rice in which the Aberdeen lab had a key role to play (de la Calle et al. 2011).

Additionally to all these biological analysis, environmental samples were also analysed, which were in an archaeological context. The measurements using the ultra-trace element concentration in peat bogs made it possible to determine whether or not ores were excavated in a large scale in antiquity (Meharg et al. 2012).
Exploitation Route The food standard agencies may use this facility in their screening analysis of the food basket in the UK, or SMEs in the food industry may want to control their food production with regards to the toxic element levels. Additionally the food industry is increasingly interested in the selenium addition to their produce. Since the molecular form of selenium is important for its health effects, it is important to identify how fortified food commodities incorporate additional selenium. The analytical facilities established in Aberdeen are the perfect instrument to identify those small concentrations of essential molecular forms of metals and metalloids. The research of trace elements in biological and environmental samples is important in different fields of science but bridges over to social science. This becomes currently apparent when the fundamental research conducted using the instrument ICP-MS, helps to define the safe limits of toxic elements such as arsenic in foodstuff such as rice. The performance of the analytical laboratory is instrumental to set the correct value for the trace amounts of an undesired metal in food commodities.

Here this sophisticated instrument is used to validate methodologies which utilises cheaper instrumentations, since the HR-ICPMS, acquired on this grant can measure the trace elements interference free. This is the reason why this lab has been used as an expert lab to characterise a sample before it has been send out to more than 100 laboratories world-wide.
Sectors Agriculture, Food and Drink,Chemicals,Environment

Description The additional high resolution ICP-MS bought from this instrumental grant accomplished a UK wide unique facility in which an HPLC is coupled online simultaneously to an elemental mass spectrometer (ICP-MS) and the molecular mass spectrometer (Orbitrap) for qualitative and quantitative determinations of molecules containing a heteroatom without any standards available.
First Year Of Impact 2009
Sector Agriculture, Food and Drink,Chemicals,Environment