Abstract
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.
Highlights
Recent reports indicate that it is not always possible to reliably extrapolate from changes in mRNA expression levels to changes in protein expression [1]
Isotope-coded affinity tag (ICAT)1 reagents were used in combination with ion trap (IT) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to study p53-dependent cell death processes that occur in primary mouse cortical neurons after DNA damage [3, 4]
We report here the use of mass spectrometry and differential isotope labeling to quantitate simultaneous changes in the expression of hundreds of neuronal proteins after DNA damage induced by exposure to the topoisomerase I inhibitor, camptothecin
Summary
Preparation of Neuronal Cell Cultures and Transfection—Primary cortical neuronal cultures were established from wild-type (p53ϩ/ϩ) newborn pups (day 0) and from a p53 knock-out (p53Ϫ/Ϫ) strain (C57BL/ 6 ϫ 129 SV background) as described previously [4, 6]. The labeled cDNA probes from each sample of control- and camptothecin-treated neurons were combined and hybridized at 42 °C in a humidified chamber for 14 –16 h to glass slides (Amersham Biosciences) that had been spotted with the 15K mouse unigene set (obtained from NIA, National Institutes of Health). Each expressed sequence tag was represented in duplicate on the slide set Both Cy3-labeled and Cy5-labeled cDNA probes were prepared from each sample and used for hybridization in reversed combinations to control for artifactual biases in fluorescence intensity ratios resulting from differences inherent to the Cy3 and Cy5 dyes. Expression ratios of 1.4 or greater were considered significantly different
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