Proteome analysis of DNA damage-induced neuronal death using high throughput mass spectrometry.
Isotope-coded affinity tag reagents and high throughput mass spectrometry were used to quantitate changes in the expression of 150 proteins in mouse wild-type (p53(+/+)) cortical neurons undergoing DNA damage-induced death. Immunological techniques confirmed several of the changes in protein expression, but microarray analysis indicated that many of these changes were not accompanied by altered mRNA expression. Proteome analysis revealed perturbations in mitochondrial function, free radical production, and neuritogenesis that were not observed in p53-deficient neurons. Changes in Tau, cofilin, and other proteins recapitulated abnormalities observed in neurodegenerative states in vivo. Additionally, DNA damage caused a p53-dependent decrease in expression of members of the protein kinase A (PKA) signaling pathway. PKA inhibition promoted death in the absence of DNA damage, revealing a novel mechanism by which endogenous down-regulation of PKA signaling may contribute to p53-dependent neuronal death. These data demonstrate the power of high throughput mass spectrometry for quantitative analysis of the neuronal proteome.
Johnson
Mark D
MD
Department of Neurological Surgery, University of Washington School of Medicine, Seattle, Washington 98195-6470, USA.
Yu
Li-Rong
LR
Conrads
Thomas P
TP
Kinoshita
Yoshito
Y
Uo
Takuma
T
Matthews
Jesse D
JD
Lee
Sang-Won
SW
Smith
Richard D
RD
Veenstra
Timothy D
TD
Morrison
Richard S
RS
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D004396:Coloring Agents / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004249:DNA Damage; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D005609:Free Radicals; D016158:Genes, p53; D015151:Immunoblotting; D007150:Immunohistochemistry; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D020411:Oligonucleotide Array Sequence Analysis; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010590:Phalloidine / Q000494:pharmacology; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D004396:Coloring Agents; D018076:DNA, Complementary; D005609:Free Radicals; D010455:Peptides; D020543:Proteome; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D010590:Phalloidine; D017868:Cyclic AMP-Dependent Protein Kinases; D010100:Oxygen