Lipid metabolism is complex and involves many metabolic reactions resulting in an enormous number and variety of lipid entities within living cells. LIPID MAPS (http://www.lipidmaps.org/) currently stores more than 40000 in-silico lipid structures. Wide dynamic range of lipid concentrations can vary by six orders 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.
In order to compare lipidomic data absolute quantitative data must be compared as relative values can vary widely between laboratories and instruments due to factors including analyst errors, sample preparation method differences (e.g. extraction methods) and ion suppression effects etc. The lack of accurate characterisation of the lipid species also severely hinders interpretation of disease and physiological states.
The proposed platform solution is an integrated high throughput analytical tool for accurate and robust measurement (>1500 injections) of a carefully selected set of targeted lipid species (~currently 500) from sample preparation through to data handling and pathway elucidation. In depth targeting of specific class of lipids was built in. Validation of the chromatographic method was conducted at multiple sites (Wilmslow, Beverly and Duke University) by different analysts, showing robustness and ease of method transfer. A rapid total lipid extraction method (IPA) and a MTBE extraction protocol for plasma are under development and show promise allowing sample flexibility.
The calibration, system suitability and QC standards were sourced pre-mixed from commercial vendors (Avanti Lipids). SymphonyTM Software automated the entire workflow and integrated with Skyline (MacCoss Lab Software (https://skyline.ms/project/home/begin.view) for data processing enhancing efficiency and flexibility. Pathway mapping tools and discovery tools by XCMS/METLIN/Metaboanalyst can also be confidently applied to determine biological relevance of changes in concentration and make data comparisons between laboratories.