Global investigation of p53-induced apoptosis through quantitative proteomic profiling using comparative amino acid-coded tagging.
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abstract
p53-induced apoptosis plays a pivotal role in the suppression of tumorigenesis, and mutations in p53 have been found in more than 50% of human tumors. By comparing the proteome of a human colorectal cancer cell transfected with inducible p53 (DLD-1.p53) with that of the control DLD-1 cell line using amino acid-coded mass tagging (AACT)-assisted mass spectrometry, we have broadly identified proteins that are upregulated at the execution stage of the p53-mediated apoptosis. In cell culturing, the deuterium-labeled (heavy) amino acids were incorporated into the proteome of the induced DLD-1.p53 cells, whereas the DLD-1.vector cells were grown in the unlabeled medium. In high-throughput LC-ESI-MS/MS analyses, the AACT-containing peptides were paired with their unlabeled counterparts, and their relative spectral intensities, reflecting the differential protein expression, were quantified. In addition, our novel AACT-MS method utilized a number of different heavy amino acids as internal markers that significantly increased the peptide sequence coverage for both quantitation and identification purposes. As a result, we were able to identify differentially regulated protein isozymes that would be difficult to distinguish by ICAT-MS methods and to obtain a large dataset of the proteins with altered expression in the late stage of p53-induced apoptosis. The regulated proteins we identified are associated with several distinct functional categories: cell cycle arrest and p53 binding, protein chaperoning, plasma membrane dynamics, stress response, antioxidant enzymes, and anaerobic glycolysis. This result suggests that the p53-induced apoptosis involves the systematic activation of multiple pathways that are glycolysis-relevant, energy-dependent, oxidative stress-mediated, and possibly mediated through interorganelle crosstalks.
