Proteomic analysis of in vivo phosphorylated synaptic proteins
Proteomic analysis of in vivo phosphorylated synaptic proteins
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DOI：
10.1074/jbc.m411220200
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发表时间：
2005-02-18
影响因子：
4.8
通讯作者：
Grant, SGN
中科院分区：
文献类型：
--
作者：
Collins, MO;Yu, L;Grant, SGN
在神经系统中，蛋白质磷酸化是突触功能的基本特征。虽然已知蛋白磷酸化对于许多突触过程和疾病是重要的，但对突触蛋白的整体磷酸化知之甚少。杂合性和低丰度使得蛋白磷酸化分析困难，特别是对于哺乳动物组织样品。使用一种新的方法，结合蛋白质和肽固定金属亲和色谱和质谱数据采集策略，我们已经产生了第一个大规模的小鼠突触磷酸化蛋白质组图。我们报告了650多个磷酸化事件，对应于331个位点（289个已明确分配），其中92%是新的。这些代表了79种蛋白质，其中一半是新的磷蛋白，包括几种高度磷酸化的蛋白质，如MAP1B（33个位点）和巴松管（30个位点）。另外149个候选磷蛋白通过分析蛋白质固定化金属亲和色谱富集的组成来鉴定。观察到所有主要的突触蛋白类，包括重要的突触前和突触后复合物以及低丰度信号蛋白的组分。肽阵列的生物信息学和体外磷酸化测定表明，少数激酶磷酸化许多蛋白质，并且每个底物被许多激酶磷酸化。这些数据大大增加了突触蛋白磷酸化的现有知识，并支持一个模型，突触磷酸化蛋白质组在功能上组织成一个高度互连的信号网络。
In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.