Global investigation of p53-induced apoptosis through quantitative proteomic profiling using comparative amino acid-coded tagging.
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.
Gu
Sheng
S
MS M888, B-2, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Liu
Zhihe
Z
Pan
Songqin
S
Jiang
Zeyu
Z
Lu
Huimei
H
Amit
Or
O
Bradbury
E Morton
EM
Hu
Chien-An A
CA
Chen
Xian
X
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D015179:Colorectal Neoplasms / Q000235:genetics; D021984:Cyclophilin A / Q000737:chemistry; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000737:chemistry; D003903:Deuterium / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology*; D015854:Up-Regulation
D000596:Amino Acids; D000081406:Peptidyl-Prolyl Isomerase F; D011506:Proteins; D016159:Tumor Suppressor Protein p53; C081786:cyclophilin B; D003903:Deuterium; D021984:Cyclophilin A; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase