Study of neurotrophin-3 signaling in primary cultured neurons using multiplex stable isotope labeling with amino acids in cell culture.
Study of neurotrophin-3 signaling in primary cultured neurons using multiplex stable isotope labeling with amino acids in cell culture.
复制标题
使用细胞培养物中氨基酸的多重稳定同位素标记研究原代培养神经元中的神经营养素 3 信号传导。
DOI：
10.1021/pr200016n
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发表时间：
2011
影响因子：
4.4
通讯作者：
Neubert,ThomasA
中科院分区：
文献类型：
--
作者：
Zhang,Guoan;Deinhardt,Katrin;Chao,MosesV;Neubert,ThomasA
在细胞培养物中用氨基酸进行的常规稳定同位素标记（SILAC）需要蛋白质的广泛代谢标记，因此难以应用于在SILAC培养物中不分裂或不稳定的细胞。使用两组不同的重氨基酸进行标记允许使用部分标记的细胞进行简单的SILAC定量，因为两个细胞群体总是被同等地标记。在这里，我们报告的应用程序，这种标记策略的原代培养的神经元。我们证明了蛋白质定量不受神经元蛋白质不完全标记的影响。我们用这种方法来研究神经营养素-3（NT-3）信号在原代培养的神经元。令人惊讶的是，我们的研究结果表明TrkB信号是由NT-3在皮层神经元中诱导的信号网络的主要组成部分。此外，参与蛋白质，如VAMP 2，Scamp 1，和Scamp 3表明，NT-3可能会导致突触囊泡的胞吐增强。
Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive metabolic labeling of proteins and therefore is difficult to apply to cells that do not divide or are unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. Here we report the application of this labeling strategy to primary cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement of proteins such as VAMP2, Scamp1, and Scamp3 suggests that NT-3 may lead to enhanced exocytosis of synaptic vesicles.