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Metabolomics: defining the standards for sample preparation of small molecules

Final Report Summary - METABOLOMICSSTANDARD (Metabolomics: defining the standards for sample preparation of small molecules)

Metabolomics is the latest of the -omics technologies that concerns the measurement of the metabolome. Like other omic technologies, metabolomics aims at the comprehensive profiling of target materials. It is generally defined as both the qualitative and quantitative analysis of all metabolites in an organism. By measuring the three-omes: transcriptome, proteome and metabolome of an organism under different conditions, one may be able to understand the function of individual genes. This could be the effect of a medicine on a patient, test animal or cell; the changes caused by a disease and identify markers for a rapid diagnosis; the defense of a plant against insects or microorganisms; etc.

To measure all metabolites in an organism is a major task as it concerns a large number of compounds with very different physical characteristics which are present in very different amounts and thereby makes the analysis of the whole metabolome both qualitatively and quantitatively a major challenge. Development of metabolomics is driven by the functional genomics but there are many other fields of application, both commercial and scientific.

As the youngest of the -omics metabolomics, this scientific field is still developing. One of the major constraints is the lack of a public database for metabolomic data like they exist now for gene and protein sequences and for proteomics. The great strength of molecular biology is the availability of gene sequence databases that allow the storage of data in an 'eternal' way and thus makes that the data can be used by all researchers. An important hurdle here is lack of standardisation in the methods applied for the metabolome analysis. Many steps are needed in metabolomic analysis. These include sample preparation, extraction, chemical and statistical analysis, and bioinformatics. Although none of the steps can be ignored, the first part of metabolomic experiments, i.e. the pre-analytical steps such as sample preparation and extraction, have not gained the same attention as the development of chemical or statistical analysis. However, sample preparation and extraction are the key steps to preserve the metabolites as they exist in the organisms. Therefore, standardisation of the pre-analytical steps is not only a must to be able to build up public metabolomic databases but should also be considered of importance in every metabolomics experiment.

The METABOLOMICSSTANDARD project focused on standardisation in the methods applied for the metabolome analysis and was build upon the expertise of Qiagen in developing standard methods for sample preparation in life sciences, and the extensive knowledge on natural products isolation and analysis of the section metabolomics at the Leiden University. The project aimed at establishment of standardise d protocols for the sample preparation for metabolome analysis in different biological materials, including the specifications of these methods, such as critical steps, reproducibility, and standard deviations. Focus was on the determination of a model metabolomics workflow for a model target material and the search of suitable stabilisation, fractionation and extraction agents and methodologies.

We started with a search for potentially stabilising agents. 11 inorganic substances were taken into consideration for further investigation. An enzyme model assay was developed for screening their potential to reduce metabolic activity. Further, a suitable model for setting up a model standard workflow was established. An extensive literature study of the extraction processes in metabolomic analysis was performed and it was decided to narrow the methodology focus on nuclear magnetic resonance (NMR) and Liquid chromatography mass spectrometry (LCMS) methodologies. A further screening for potential stabilising agents that where compatible with the chosen analytical techniques was performed and three mixtures of stabilising agents were taken to assess the impact of the selected stabilisation solutions. Studies were carried out for plant material as well as human urine and blood. Fresh human blood plasma was analyzed for its robustness against the stabilisation solutions. A stabilisation strategy was established which is based on four principles:

(1) pH buffering;
(2) redox stabilisation;
(3) enzyme inhibition; and
(4) fast separation of molecules based on their molecular size.

This more generic strategy has the advantage to be suitable for different classes of compounds. We further established two devices that support the fast stabilisation approach. Finally, the feasibility of the developed protocols for sample preparation for a subsequent metabolome analysis with NMR was shown.

The knowledge generated within the project has been shared with researchers of the participating partners and further disseminated via different channels to the scientific community such as talks and publications. In 2012, a Leiden meeting 'From metabolic profiling to metabolomics' was organise d. In 20 short lectures, the development of metabolomics was shown by researchers working in the natural products field in the past decades. This symposium clearly showed that the methods have been around already for many years, but that real integration is not going as fast as desired. Already in the early stages of gas chromatography more than 40 years ago metabolic profiling was used, but only the past ten years highly standardise d protocols have been generally accepted as a basis for true metabolomics applied in particularly a systems biology type of approach to study various biological systems. Also hands-on courses were given, in which the participants had the possibility to practice the protocols used in the pre-analytical part of metabolomics, i.e. the immediate quenching of metabolism, grinding and extraction of the biological samples. Also the participants were guided in applying multivariate data analysis of metabolomics data sets. These hands-on courses were highly appreciated by the participants.
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Metabolomics is the latest of the -omics technologies that concerns the measurement of the metabolome. Like other omic technologies metabolomics aims at the comprehensive profiling of target materials. It is generally defined as both the qualitative and quantitative analysis of all metabolites in an organism. By measuring the three-omes: transcriptome, proteome and metabolome of an organism under different conditions, one may be able to understand the function of individual genes. This could be the effect of a medicine on a patient, test animal or cell; the changes caused by a disease and identify markers for a rapid diagnosis; the defense of a plant against insects or microorganisms; etc. Within the second half of the project the focus was on the refinement of the stabilisation strategy that was introduced in period one. First, the single elements of the strategy were examined and improved. Later, the feasibility of the combination of all elements was tested and the protocols adapted accordingly. The feasibility of the combined elements with the chosen sample material and the downstream analytical method has been tested. A market analysis has been performed to assess the market needs for metabolomics sample stabilisation. Information regarding the specific gaps in current sample preparation protocols has been collected and analyzed. In addition to that, also a market entry strategy has been developed and the market volume has been estimated. According to the market study two different sample preparation protocols have been established together with corresponding sample preparation devices that need only minimal user interaction during sample collection, storage and shipment. The knowledge generated within the project has been shared with researchers of the participating partners and further disseminated via different channels such as talks and publications to the scientific community. In 2012, a Leiden meeting 'From metabolic profiling to metabolomics' was organise d. In 20 short lectures, the development of metabolomics was shown by researchers working in the natural products field in the past decades. Also hands-on courses were given, in which the participants had the possibility to practice the protocols used in the preanalytical part of metabolomics, i.e. the immediate quenching of metabolism, grinding and extraction of the biological samples. These hands-on courses were highly appreciated by the participants.