Abstract
Lipid metabolism is complex and involves a large number of metabolic reactions resulting in an enormous number and variety of actual lipids within living cells. LIPIDMAPS currently stores more than 40000 lipid structures. The dynamic range of lipid concentrations in biological systems can vary by 106 or more (from nanomolar fatty acids to attomolar eicosanoid lipid mediators). The level of precision of most systems-wide measurements is not yet sufficient to detail specific levels or concentrations of cellular components. Comparing lipidomic data across laboratories requires absolute quantification since relative values can vary widely not only between laboratories and between instruments due to various factors including: analyst errors, sample preparation differences (e.g. extraction methods) and ion suppression when using ESI MS. The lack of accurate characterisation of the lipid species also severely hinders interpretation of lipid metabolism associated with disease and physiological states. The proposed platform involves integrated high throughput analytical tool for accurate and robust measurement (>1500 injections) of lipid from sample preparation through to data handling and pathway elucidation. The platform can also be used for more in depth targeting of specific class of lipids of interest. Validation of the chromatographic method is performed at multiple sites by different analysts to show robustness and ease of method transfer. A rapid total lipid extraction method involving IPA and MTBE for plasma shows promising results. This will grant researchers more flexibility depending on their specific needs and requirements. The calibration, system suitability and QC standards used in this platform is sourced pre-mixed from commercial vendors (Avanti Lipids). SymphonyTM Software is used to automate the entire workflow and integrated with Skyline for data processing to enhance efficiency and flexibility. Once quantitative data has been generated and processed, pathway mapping tools can be used to determine the biological relevance of changes in concentration, and make data comparisons between laboratories.