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Please use this identifier to cite or link to this item: http://hdl.handle.net/1807/27374

Title: Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p Interactomes
Authors: Wheaton, Sarah
Advisor: Raught, Brian
Department: Medical Biophysics
Keywords: Ubiquitin-like Proteins
Affinity Purification-Mass Spectrometry
Issue Date: 31-May-2011
Abstract: The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Modification of a protein with a Ubl can alter its localization, activity and/or half-life. SUMO and Rub1p/Nedd8 are two Ubls that play important roles in a number of critical cellular processes, yet their specific cellular functions remain poorly understood. To better understand these important Ubls, we developed a robust affinity purification-mass spectrometry (AP-MS) technique to generate protein-protein interaction maps for the Ubl systems. Each bait was systematically expressed as a C-terminal HA-tagged fusion protein in S. cerevisiae. A standardized method in which affinity purification via the HA epitope, followed by mild washing and mass spectrometric analysis, was performed and the data generated were used to build interaction maps. Affinity purification of the Rub1p E3 ligase Dcn1p identified a novel interaction with the AAA ATPase Cdc48p. This interaction was further studied to determine its biological significance.
URI: http://hdl.handle.net/1807/27374
Appears in Collections:Master

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