Development of Mass Spectrometry Methods for Analysis of Lipids in Mammalian and Plant Tissues

Leelyn Chong, Purdue University

Abstract

Breast cancer is one of the leading causes of cancer-related deaths in women. To date, there has yet to be an efficient diagnostic technique established for detecting the disease. Since the cellular membrane undergoes dynamic changes during tumor progression, developing a facile method that examines the changes in the major components such as lipids that comprise of the cellular bio-membrane may be useful. In mammalian cells, fatty acids (FAs) and phospholipids (PLs) are one of the predominant lipids. No studies have yet investigated isomeric lipid changes in chronic diseases such as breast cancer. Therefore, PB-MS/MS was employed to investigate the feasibility of it to determine and differentiate isomeric lipids that could be associated with breast cancer. The methodology was further applied to a disease of the central nervous system, multiple sclerosis (MSc). MSc is a chronic, autoimmune disease that damages the myelin sheath of the central nervous system. The dysregulation of lipid pathways has been implicated for MSc. Currently, there is no cure for MSc and the search for pharmacological targets of MSc is still undergoing. A shotgun lipid analysis was performed for the rapid profiling of lipids in healthy and EAE (experimental animal model for human MSc) mouse tissues, followed by measuring lipid isomeric ratios that may be associated with MSc. Lipid markers that may have potential impact on diagnosis of MSc were investigated. Moreover, the significance of unsaturated lipid isomers to MSc were determined. Besides applying the methodology in chronic diseases, plant tissues were subjected to the technique as well to study the lipids of plants in cold stress response. Plants have to deal with constant stresses that occurred in their living environment in order to sustain and survive. One type of stresses that plants are being exposed to in nature is cold stress. Not every plant can tolerate the cold and survive; thus the ability of plants to survive freezing temperatures must vary among plant species. There are several fundamental strategies that plants utilize to resist temperature stress and one of them is by altering their lipid composition in the membrane. Despite what is known, lipid regulation under cold stress in plants still requires further studies since plant lipidomics is an intricate field. Furthermore, unsaturated lipids and their structural isomers were often overlooked since there was not a feasible method established yet to detect them. Therefore, PB-MS/MS was implemented to identify lipids that may potential involvement in cold stress and subsequently distinguish C=C location isomers of these lipids in plants. Lastly, the technique was applied to perform a lipidomics study in two-dimensional (2D) and three-dimensional (3D) ovarian cancer cell cultures. For many years, 2D cell culture model has been profoundly employed in in-vitro research. 2D in vitro model systems do not accurately mimic the organismal body since tissues in the living organisms are 3D. Using a 2D cell culture model in research could slow down discoveries and lead to late-stage clinical failures. To better illustrate the essence of 3D cell culture model, this study was conducted to examine the lipid differences and essentially isomeric changes of lipids in the 2D and 3D cell culture models. The application of PB-MS/MS in these various biological systems has demonstrated its robustness and effectiveness in studying lipidomics via a shotgun lipidomics approach. Therefore, it is easy-to-use, cost effective, and less time-consuming than other techniques. Most importantly, it not only provides a sketch of the lipidome of the biological system rapidly but it also enables the simultaneous C=C characterization and quantitation of unsaturated lipids in biological specimens. Therefore, it is a tool with great potential for studying correlations of C=C lipid isomer ratios in diseases relevant to human health and nutrition well as in the plant lipid biology which is crucial to the agricultural industry. (Abstract shortened by ProQuest.)

Degree

Ph.D.

Advisors

Dai, Purdue University.

Subject Area

Analytical chemistry

COinS