Large-scale quantitative proteomic study of PUMA-induced apoptosis using two-dimensional liquid chromatography-mass spectrometry coupled with amino acid-coded mass tagging.
By coupling two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) with amino acid-coded mass tagging (AACT), we have greatly increased the analytical throughput and sequence coverage of MS-based methods for proteome-wide quantitation. The dynamic range and reproducibility of this 2D-LC-AACT quantitative approach were evaluated by profiling the mixtures with different ratios of E. coli cells grown in either regular or AACT medium. A SQL-based high thoughput MASCOT data analysis tool was developed for proteomic data sorting and mining. We investigated the early stage of apoptosis by inducing the p53 upregulated modulator of apoptosis (PUMA) through the analyses of the relative ratios of the pairwise isotope signals that were originated from the control and labeled PUMA-induced cells. In 20-hour 2D-LC-MS/MS run, 480 proteins were conclusively identified, and more than half of them were quantified. A noteworthy change in the quantitative profile was that histones and a ubiquitin conjugate protein UBC9, which are involved in DNA double-strand break (DSB) repair were significantly down-regulated in the PUMA-overexpressing apoptotic cells, suggesting the detection of DSB in the apoptotic process. The quantitative profiling efficiency of this approach was compared with the gel-based quantitative analysis scheme.
Gu
Sheng
S
MS M888, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Du
Yuchun
Y
Chen
Jin
J
Liu
Zhihe
Z
Bradbury
E Morton
EM
Hu
Chien-An A
CA
Chen
Xian
X
D000596:Amino Acids; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D015179:Colorectal Neoplasms / Q000473:pathology; D004249:DNA Damage; D003903:Deuterium; D004926:Escherichia coli / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D013058:Mass Spectrometry; D015335:Molecular Probes; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000494:pharmacology*
D000596:Amino Acids; D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D015335:Molecular Probes; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D003903:Deuterium