Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum
Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum
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DOI：
10.1016/j.jneumeth.2012.10.003
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发表时间：
2013-03-15
影响因子：
3
通讯作者：
Nothwang, Hans Gerd
中科院分区：
文献类型：
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作者：
Schindler, Jens;Ye, Juanying;Nothwang, Hans Gerd
小脑中的神经元加工涉及各种质膜蛋白如AMPA或NMDA受体的磷酸化和去磷酸化。尽管磷酸化模式的变化很重要，但尚未进行全局磷酸化蛋白质组分析。由于质膜蛋白是信号级联的主要目标，我们开发了一个协议，从一个单一的小鼠小脑开始监测其磷酸化状态。采用高分子水溶液两相体系富集质膜蛋白。随后，磷酸钙沉淀，固定化金属亲和色谱，和TiO 2相结合的顺序提取程序之前，质谱分析。这一策略导致在507种不同蛋白质中鉴定出1501个不同的天然磷酸化位点。这些磷酸化位点中有765个（51%）被定位，置信水平为99%或更高。41.4%的蛋白质定位于质膜，约一半的磷酸化位点以前没有报道过。对12个共有序列的生物信息学筛选确定了642个磷酸化位点的推定激酶。总之，在这里部署的协议确定了几百个新的小脑蛋白磷酸化位点。此外，它提供了一个有价值的工具来监测质膜蛋白质组从任何小的脑样品的兴趣在不同的生理或病理生理条件下。(c)2012 Elsevier B.V.保留所有权利。
Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane proteins are major targets of the signalling cascades, we developed a protocol to monitor their phosphorylation state starting from a single mouse cerebellum. An aqueous polymer two-phase system was used to enrich for plasma membrane proteins. Subsequently, calcium phosphate precipitation, immobilized metal affinity chromatography, and TiO2 were combined to a sequential extraction procedure prior to mass spectrometric analyses. This strategy resulted in the identification of 1501 different native phosphorylation sites in 507 different proteins. 765 (51%) of these phosphorylation sites were localized with a confidence level of 99% or higher. 41.4% of the identified proteins were allocated to the plasma membrane and about half of the phosphorylation sites have not been reported previously. A bioinformatic screen for 12 consensus sequences identified putative kinases for 642 phosphorylation sites. In summary, the protocol deployed here identified several hundred novel phosphorylation sites of cerebellar proteins. Furthermore, it provides a valuable tool to monitor the plasma membrane proteome from any small brain samples of interest under differing physiological or pathophysiological conditions. (c) 2012 Elsevier B.V. All rights reserved.