Global investigation of p53-induced apoptosis through quantitative proteomic profiling using comparative amino acid-coded tagging
Global investigation of p53-induced apoptosis through quantitative proteomic profiling using comparative amino acid-coded tagging
复制标题
DOI：
10.1074/mcp.m400033-mcp200
复制
发表时间：
2004-10-01
影响因子：
7
通讯作者：
Chen, X
中科院分区：
文献类型：
--
作者：
Gu, S;Liu, ZH;Chen, X
p53诱导的细胞凋亡在抑制肿瘤发生中起关键作用，在50%以上的人类肿瘤中发现p53突变。通过使用氨基酸编码质量标记（AACT）辅助质谱法比较转染诱导型p53 （DLD-1.p53）的人结直肠癌细胞的蛋白质组与对照DLD-1细胞系的蛋白质组，我们大致确定了在p53介导的凋亡执行阶段上调的蛋白质。在细胞培养中，氘标记（重）氨基酸被整合到诱导的DLD-1的蛋白质组中。p53细胞，而DLD-1。载体细胞在未标记的培养基中生长。在高通量LC-ESI-MS/MS分析中，将含有aact的肽与未标记的肽配对，并对其相对光谱强度进行量化，以反映差异蛋白的表达。此外，我们的新型AACT-MS方法利用许多不同的重氨基酸作为内部标记，显著增加了定量和鉴定目的的肽序列覆盖率。因此，我们能够鉴定出ICAT-MS方法难以区分的差异调节的蛋白同工酶，并获得了p53诱导的细胞凋亡晚期表达改变的蛋白质的大型数据集。我们发现的受调节蛋白与几个不同的功能类别有关：细胞周期阻滞和p53结合、蛋白质伴侣、质膜动力学、应激反应、抗氧化酶和厌氧糖酵解。这一结果表明p53诱导的细胞凋亡涉及糖酵解相关、能量依赖、氧化应激介导的多种途径的系统激活，并可能通过细胞器间串扰介导。
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.