Development and application of mass spectrometry based proteomics technologies to decipher ku70 functions

Abstract

Ku70 is a protein with multiple biological functions. Given the dual role of Ku70 in DNA repair and apoptosis, it might be a therapeutic target for cancer treatment. We employed epitope-tag purification and mass spectrometry based proteomic methods to characterize the Ku70 protein complex to unravel its biological functions. We also developed a novel effective peptide purification and concentration method for in-gel sample. The pulled-down complex was analyzed by both SDS-PAGE coupled to MALDI-TOF/TOF-MS and shotgun LC-MS/MS proteomics approaches. Consequently, 151 proteins were characterized in Ku70 protein complexes. These proteins play a diverse range of biological functions from DNA repair to transcriptional regulation to cellular signal mediator. As Ku70 acetylation has been reported to be a pivotal post translational modification that regulates Ku70 activities, we finally identified the potential acetylation sites of Ku70 by both in vivo and in vitro acetylation analysis.