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Molecular & Cellular Proteomics 7:1067-1076, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Stable Isotopic Labeling by Amino Acids in Cultured Primary Neurons

Application to Brain-derived Neurotrophic Factor-dependent Phosphotyrosine-associated Signaling ^*,S

Daniel S. Spellman, Katrin Deinhardt^,, Costel C. Darie, Moses V. Chao^, and Thomas A. Neubert^,¶,||

From the ^¶ Department of Pharmacology, Departments of Cell Biology, Physiology and Neuroscience, and Psychiatry, and Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York 10016

Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we demonstrated that stable isotope labeling by amino acids in cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by brain-derived neurotrophic factor (BDNF), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitations comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase for BDNF, and others such as hepatocyte growth factor-regulated tyrosine kinase substrate and signal-transducing adaptor molecule, which are proteins known to regulate intracellular trafficking of receptor tyrosine kinases. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics.

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