The Purification and Identification of Interactors to Elucidate Novel Connections in the HEK 293 Cell Line

• Main Author: Hawley, Brett
• Other Authors: Figeys, Daniel
• Language: en
• Published: Université d'Ottawa / University of Ottawa 2012
• Subjects: Proteomics; Alzheimer's Disease; Apolipoprotein E; Clusterin; PICALM; Synuclein; Low-density lipoprotein receptor; Platelet activating factor receptor; affinity purification
• Online Access: http://hdl.handle.net/10393/23536; http://dx.doi.org/10.20381/ruor-6225

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.