Lipidomic Profiling of Autophagosome Membrane by Mass Spectrometry

Autophagy is molecular machinery for “self-eating” in cells. It is a highly conserved process of self-degradation of cellular components in response to extra or intracellular stress and signals such as starvation, growth factor deprivation, and pathogen infection. This self-digestion not only provides nutrients to maintain vital cellular functions during fasting but also can rid the cell of superfluous or damaged organelles, misfolded proteins, and invading micro- organisms. Autophagy is a unique membrane trafficking process whereby newly formed membranes, termed phagophores, engulf parts of the cytoplasm leading to the production of double-membraned autophagosomes that get delivered to lysosomes for degradation. The origin of the autophagosomal membrane and how its formation is initiated remain open questions after more than 50 years of investigation. It is still not well understood how the membranes grow and expand to form the autophagosome. In this project, we are using Liquid Chromatography – Mass Spectrometry (LC-MS) to elucidate lipid composition of autophagosome. HeLa-Difluo™ hLC3 cells containing green fluorescence protein (GFP) fused with autophagy protein LC3B are treated with rapamycin for four hours to stimulate autophagy. During the last two hours of treatment period, Bafilomycin A1 are added to block the fusion of autophagosome and lysosome. Cells are then harvested. Autophagosome are purified by immunoprecipitation using GFG antibody. Lipids are extracted and analyzed by LC-MS. The results of this project will contribute to the complete elucidation of lipid composition of the autophagosome membrane and the role of lipids in autophagy.