Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum
Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum
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DOI：
10.1074/mcp.d500009-mcp200
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发表时间：
2006-06-01
影响因子：
7
通讯作者：
Peng, Junmin
中科院分区：
文献类型：
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作者：
Cheng, Dongmei;Hoogenraad, Casper C.;Peng, Junmin
中枢兴奋性突触的突触后密度（PSD）对突触后信号传递至关重要，其组成成分在不同的神经元亚型和脑结构中存在异质性。在这里，我们报道了从成年大鼠前脑和小脑纯化的psd中蛋白质的大规模相对和绝对定量。采用可切割ICAT策略和LC-MS/MS测定PSD蛋白谱。共鉴定并定量了296个蛋白，其中43个蛋白在前脑和小脑之间的丰度变化具有统计学意义，表明psd在不同脑区之间存在明显的分子异质性。此外，我们采用绝对定量策略，以合成的同位素标记肽为内标，测量了前脑和小脑中32个关键PSD蛋白的摩尔丰度。这些数据证实了钙/钙调素依赖性蛋白激酶II和PSD-95的丰度，并揭示了PSD中谷氨酸受体、支架蛋白和信号分子之间意想不到的化学计量比。我们的数据还表明，绝对定量方法非常适合于靶向定量蛋白质组学分析。总的来说，本研究描述了前脑和小脑PSD之间的关键分子差异，并为测量PSD的分子化学计量学提供了定量框架。
The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD.