Development of a method for isolating pure endoplasmic reticulum (ER) and characterizing its molecular components

ER is a metabolic compartment that is critical for protein, lipid, and glucose metabolism, as well as calcium homeostasis. It consists of a network of branching tubules extending from the outer membrane of the nuclear envelope to the plasma membrane. Therefore, it has a large and continuous lumen, which occupies more than 10% of the cell volume. The luminal micro-environment is characteristically different from the cytosol. Besides the presence of ER-related human syndromes, ER dysfunction is associated with a range of neurological disorders, such as multiple sclerosis, Parkinson's, and Huntington's diseases, but the pathogenic mechanisms remain obscure. To allow the study of the ER metabolism in health and diseases, our lab aims to develop a method utilizing magnetic immunopurification (IP) of HA-tagged ER to achieve rapid isolation of pure and intact ER that is compatible with subsequent analysis of their content. By using LC-MS, we can establish a comprehensive map of the human ER proteome, metabolome, and lipidome.