Investigation of Biological Membranes by NMR and ESI-MS Methodologies

In the last decade there has been a new reappraisal of the function of lipids in the cell life, not only for their structural role as cell wall or for their energy storage function, but also for their significant function on signalling and protein recognition processes. This new attention on lipids has led to a new research field in the metabolomics world called “Lipidomics”. Lipidomics is more than just the complete characterization of all lipids in a particular biological sample. It is the comprehensive understanding of the influence of all lipids on a biological system with respect to cell signalling, membrane architecture, transcriptional and translational modulation, cell-cell and cell-protein interaction and response to environmental changes over time. The critical role of lipids in cell, tissue and organ physiology is already demonstrated by many human diseases involving the disruption of lipid metabolic enzymes and pathways. Examples of such diseases include diabetes, cancer, neurodegenerative disorders and infectious diseases. This represents a clue for understanding the molecular diversity observed in membrane phospholipids. Subtle biophysical properties are also another possible explanation of strong interest toward lipids especially with reference to the emerging field of heterogeneous membrane microdomains (rafts). The major goal in lipidomics is the identification of metabolic pathways which are activated or deactivated during development of an organism or when a cell is shifted from an established physiological condition to another physiological or pathological condition (metabolic learning). A better understanding of the regulation of underlying metabolic pathways is necessary to design novel strategies for intervention. In this thesis attention has been focused on two biological systems to demonstrate how the study of membrane lipids can help for a better understanding of the mechanisms involved in different biochemical processes. The first system tackled by our methodology is represented by lipid components in detergent resistant membranes (DRM) associated with the expression of Prostate-specific membrane antigen (PSMA); the latter is a 750-residue type II transmembrane glycoprotein of the normal prostate cells and one of the most promising biomarkers of prostate carcinoma as its expression is drastically increased in cancer cells protein involved in the prostate carcinoma. In particular, the complex glycosylated form of the protein is found in Lubrol insoluble DRMs. Many essential cellular events, such as protein sorting, endocytosis and signal transduction pathways, are triggered via association of the proteins directly implicated in these processes with DRMs. In the present work we report on the lipid composition of PSMA-associated microdomains. A qualitative screening was made by thin layer chromatography (TLC) followed by an extended NMR analysis of the sample. In particular, by taking 1H-, 13C- ed 31P-NMR spectra we were able to detect and quantify cholesterol and the relative contribution of all the lipids belonging to a given PL class (such as PC, PE and SM) with respect to the overall PL composition. On the other hand, Electrospray Ionization (ESI-MS) measurements and in-source collisional induced dissociations (CID) carried out on the same sample allowed us not only to establish the distribution of lipids among the PL classes but also to recognize some structural features such as the length and the number unsaturations of their acyl chains. The second problem tackled by following similar analytical methodologies was to analyze the detailed membrane lipids profile of insect larvae of the species Pseudodiamesa branickii and Diamesa cinerella to obtain information about the biochemical mechanisms involved in their thermal adaptative capabilities. Through NMR and LC-ESI-MS experiments we have obtained information about the composition of the lipid pool of these larvae that is formed essentially by triglycerides and phospholipids, with a variable inter-species PC/PE ratio. Coupling LC-MS profiling methods with in-source fragmentation data further has enhanced the ability to probe the lipidome, by supplementing lipid identification with structural information. We have observed an high level of unsaturation of the fatty acid chain as expected for low temperature-adapted species since unsaturated chain promote a more disordered and flexible membrane structure.. For Diamesa cinerella, from ESI data we could also infer some possible mechanism adopted to increase unsaturation of the fatty acid chain by the action of desaturases enzymes or by the insertion of PUFA chains.