Proteomic Assays of Nicotine-induced Golgi Fragments and Changes in Glycosylation

Overview: We are performing experiments to characterize the molecular changes that occur during the process of nicotine upregulation that is tied to nicotine addiction. We recently found that nicotine alters Golgi apparatus morphology causing increases in mobile vesicles that derive from the Golgi. In one project, we will use proteomic assay to analyze the identity of proteins found in purified preparations of the Golgi vesicles using Proteomic Proximity Labeling methods to identify vesicles proteins and their interactions. The nicotine-induced changes in Golgi morphology also causes changes in complex trimming of α4β2-type nicotinic acetylcholine receptor N-linked glycans, particularly the addition of sialic acid. In the second project, LC MS/MS experiments in collaboration with the Discovery Core are underway to analyze the specific changes in N-linked glycosylation with nicotine exposure. We will be utilizing the Orbitrap Fusion along with the Byonics Software for data collection and site determination, respectively.