Abstract
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.
Highlights
Cultured primary neurons are a well established model for the study of neuronal function in vitro
To assure that SILAC could be applied to non-dividing neurons, which do not have the advantage of label dilution effects enjoyed by dividing cells, we tracked the levels of label incorporation for individual proteins and showed that the majority of proteins do achieve significant levels of stable isotope labeling
All proteins identified from whole cell lysates demonstrated significant levels of label incorporation by 14 days in vitro (Fig. 2). This analysis was limited to proteins containing at least one peptide that could be tracked through nine of the 10 time points. These results indicated that proteins from a differentiated, non-dividing cell population such as cultured primary neurons can be labeled by amino acids in culture
Summary
Cultured primary neurons are a well established model for the study of neuronal function in vitro. 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. These proteins function as survival factors that ensure a match between surviving neurons and appropriate targets during development of the mammalian nervous
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