Development of Mass Spectrometric Methods for the Quantification of Membrane Lipids : Studies on Mitochondria, T Cells, Golgi Membranes and COPI Vesicles

Abstract

Biological membranes contain more than thousand different lipid classes and lipid species that are far from being fully characterized. In order to understand molecular processes that are connected to membrane lipids a continuous development of analytical methods is required. In this thesis, nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to establish methods for the quantification of cardiolipin (CL), diacylglycerol (DAG) and the phosphoinositides PIP and PIP2. The methods were applied for the analysis of mitochondria, Golgi membranes and T cells in order to address scientific questions: I. The quantitative analysis of CL in mitochondria, isolated from yeast mutants, showed that GEP genes (genetic interactors of prohibitins) are involved in the regulation of mitochondrial phospholipids CL and phosphatidylethanolamine. Strikingly, the mass spectrometric identification of the lipid intermediate phosphatidylglycerolphosphate (PGP) in mitochondria from delta gep4 mutants supported the identification of Gep4 as a novel PGP phosphatase required for CL biosynthesis. II. The quantitative analysis of DAG revealed that ilimaquinone (IQ)-induced vesiculation of the Golgi complex significantly affects the level of DAG. This result is consistent with previous observations that lipid-modifying enzymes, such as phospholipase D and PA phosphatase, are activated and suggests that membrane lipids play a critical role during the process of IQ-mediated Golgi vesiculation. III. The quantitative analysis of PIP, PIP2 and DAG in conventional T cells showed that T cell receptor (TCR)-induced stimulation significantly affects the level of signaling lipids. This result was an important readout to address the question whether regulatory T cells interfere with proximal lipid signaling events in conventional T cells. Furthermore, a lipidome analysis of Golgi membranes and COPI vesicles was performed. The generated data confirmed previous findings that sphingomyelin and cholesterol are segregated during the formation of COPI vesicles. Moreover, the lipid analysis revealed that COPI vesicles display a lipid composition similar to the endoplasmatic reticulum, with elevated levels of phosphatidylcholine and phosphatidylinositol. The characteristic lipid composition supports the scenario that COPI vesicle formation occurs at liquid-disordered domains in the Golgi complex.