| Summary: | Recent advancements in mass spectrometry have facilitated new analytical approaches capable of comprehensively characterizing metabolites in biological samples. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) combines excellent mass accuracy (ppm<1) and ultra-high resolution, which enables the separation and identification of individual components within complex mixtures, and the determination of elemental composition for each detected mass. FTICR-MS is an ideal method for non-targeted metabolomics as the majority of small molecular compounds (100-1000 Da) in a biological sample can be detected. The objective of this research was to investigate metabolomic alterations associated with key cellular processes deemed fundamental to cancer development and progression.
Differentiating U937 cells, fibroblasts synchronously progressing through the cell cycle and a transformed cell line containing a temperature sensitive oncogene were collected and subject to FTICR-MS analysis for non-targeted comprehensive metabolomics. Putative metabolite identifications were confirmed with targeted metabolite analysis using multiple reaction monitoring triple quadrupole mass spectrometry. Analysis of the resulting metabolic profiles revealed robust metabolic alterations associated with fundamental cellular processes. Changes in glycerolipid content were observed in all cellular processes studied. During cell cycle progression, elevated levels of triacylglycerols and vinyl acylglycerols were detected as cells approached mitosis; increased levels of plasmalogens were detected during the induced differentiation of human leukemic cells and activation of the oncogene p130gag-fps in fibroblasts resulted in increased levels of phospholipids, including plasmalogens. When de novo fatty acid synthesis was inhibited in the differentiation cell model, the cells were not able to complete the differentiation process. Removal of the inhibitor resulted in increased lipid content, particularly plasmalogens, and the continuation of differentiation, suggesting a requirement for the de novo synthesis of lipids during this cellular process.
This work demonstrates the advantages of non-targeted metabolic profiling for identifying non-intuitive metabolic associations with specific cellular processes. Collectively, the results of this thesis have implicated glycerolipids, in particular phospholipids, in the processes of cell cycle progression, differentiation and tumourigenic transformation. A broadened understanding of the role of global lipid metabolism during fundamental cellular processes may one day lead to new approaches for their modulation, and potentially new therapeutic strategies.
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