|Development of mass spectrometry protocols to provide better coverage of oxidised lipidome in inflammatory disease models
|High Throughput versus High Efficiency Separations (CS)
|Dr Alpesh Thakker
Abstract Information :
Phospholipid oxidation generates a wide variety of products with potentially novel biological activities that may be associated with disease pathogenesis. To understand their role in disease requires precise information about their abundance in biological samples. Liquid chromatography-mass spectrometry (LCMS) is a sensitive technique that can provide detailed information about the oxidative lipidome, but challenges still remain.
The work in this project developed improved methods for detection of OxPLs by improvement of chromatographic separation through the comparison and optimisation of several HPLC columns such as C8, C18 and C30 reverse phase, polystyrene-divinylbenzene based monolithic, and mixed-mode hydrophilic interaction (HILIC) columns & solvent systems, with use of semi-targeted mass spectrometry approaches. The results suggest that the monolithic column was the most robust method for separating short chain oxPLs from long chain oxidised and native PLs. In addition, several approaches for method validation were explored such as testing of reproducibility and repeatability of the methods, together with the reanalysis of samples on a high resolution QToF mass spectrometer with automated quantitative data analysis using the Progenesis QI software to validate the identification. The combination of the developed methods allowed the identification of several oxPLs in biological samples. These were:
i) ascites fluid of lean and obese rat model of acute pancreatitis;
ii) isolated components of red blood cells (RBCs) infected with the malarial parasite Plasmodium falciparum; and
iii) plasma samples of healthy and diabetic patients. In addition, an evaluation of post-acquisition data handling to minimise inherent biological variation was performed. Quantitative differences in oxPLs were observed in isolated malarial components as well as other studied disease models.
Overall, several protocols were developed that provide improved performance for the identification of OxPL in biological samples that can be used as a reference method by research laboratories interested in oxidative lipidomics work.