Development and validation of a high-throughput mass spectrometric platform for the molecular characterisation of cells

Abstract

Understanding the dynamic cellular metabolism is crucial for gaining deeper knowledge of the intracellular and intercellular functions, as well as the influence of microenvironment on cells. Mass Spectrometry has been used extensively for metabolomic and lipidomic analysis of cells, especially when hyphenated with separation techniques such as liquid chromatography (LC-MS). These methods are very sensitive and can give vital information regarding the lipidome and metabolome of cells. However, the sample preparation needed for these techniques is laborious and time-consuming and does not allow for real-time analysis of cells. Laser-Desorption Rapid Evaporative Ionisation Mass Spectrometry (LD-REIMS) is an ambient ionisation technique utilising infrared (IR) lasers for the generation of molecule-rich aerosol, which can be analysed with mass spectrometry methods. This PhD thesis aims to expand the application of the automated LD-REIMS platform for the molecular characterisation of cells with minimal or no sample preparation. A systematic comparison was performed between REIMS modalities that have been used previously for cell pellet analysis – bipolar forceps and cell probe REIMS –, and the LD-REIMS autosampler, – while a protocol for the preparation-free analysis of living or frozen cells was also developed and validated. LD-REIMS outperformed other REIMS modalities in terms of classification, molecular coverage, and repeatability when used for the analysis of 5 breast and 5 colorectal cancer cell lines. The applicability of LD-REIMS was demonstrated for the analysis of cells producing biopharmaceutical compounds, where metabolic differences were demonstrated between high and low producing cell lines. LD-REIMS was also used for the detection of significant differences in the lipid metabolism of phospholipidosis-induced cells, revealing upregulation of polyunsaturated fatty acids (PUFA) and the presence of Bis(Monoacylglycero)Phosphate (BMP) lipids. In conclusion, this PhD thesis demonstrates the potential of LD-REIMS for the high-throughput analysis of cells with minimal sample preparation, and the preparation-free analysis of living cells.