Identification and Validation of Novel Spinophilin-associated Proteins in Rodent Striatum Using an Enhanced ex Vivo Shotgun Proteomics Approach
Identification and Validation of Novel Spinophilin-associated Proteins in Rodent Striatum Using an Enhanced ex Vivo Shotgun Proteomics Approach
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DOI：
10.1074/mcp.m900387-mcp200
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发表时间：
2010-06-01
影响因子：
7
通讯作者：
Colbran, Roger J.
中科院分区：
文献类型：
--
作者：
Baucum, Anthony J., II;Jalan-Sakrikar, Nidhi;Colbran, Roger J.
在发育过程中，嗜刺素通过与丝状肌动蛋白、蛋白磷酸酶1和大量其他信号蛋白的相互作用来调节兴奋性突触后功能和形态。为了深入了解亲棘素在成熟大脑中的作用，我们采用鸟枪蛋白质组学方法鉴定亲棘素免疫复合物中的蛋白质，表征了从成年啮齿动物纹状体溶解的亚细胞组分中的亲棘素相互作用体。使用小鼠亲棘素抗体产生的样品的初始分析检测到23种蛋白质，其不存在于IgG对照样品中;然而，在从亲棘素敲除组织分离的复合物中检测到这些蛋白质中的12种。使用两种不同的亲棘素抗体和敲除或IgG对照的第二次筛选鉴定了总共125种蛋白质。计算每个样品中每种蛋白质与spinophilin特异性相关的概率，并根据来自多个样品分析的概率的卡方（2）分析对蛋白质进行排序。特异性地检测到了嗜刺素和已知的相关蛋白neurabin以及蛋白磷酸酶1的多种亚型。多个，新的，spinophilin相关的蛋白质（肌球蛋白Va，钙/钙调蛋白依赖性蛋白激酶II，神经丝轻多肽，突触后密度95，α-辅肌动蛋白，和densin），然后显示相互作用的GST融合蛋白含有片段的spinophilin。另外的生物化学和转染细胞成像研究表明，α-辅肌动蛋白和致密蛋白直接相互作用的残基151-300和446-817，分别与spinophilin。两者合计，我们已经开发了一种多抗体，鸟枪蛋白质组学的方法来表征蛋白质相互作用组在天然组织中，描绘了敲除组织控制的重要性，并提供了新的见解的性质和功能的亲棘素相互作用组在成熟的纹状体。Molecular & Cellular Proteomics 9：1243-1259，2010.
Spinophilin regulates excitatory postsynaptic function and morphology during development by virtue of its interactions with filamentous actin, protein phosphatase 1, and a plethora of additional signaling proteins. To provide insight into the roles of spinophilin in mature brain, we characterized the spinophilin interactome in subcellular fractions solubilized from adult rodent striatum by using a shotgun proteomics approach to identify proteins in spinophilin immune complexes. Initial analyses of samples generated using a mouse spinophilin antibody detected 23 proteins that were not present in an IgG control sample; however, 12 of these proteins were detected in complexes isolated from spinophilin knock-out tissue. A second screen using two different spinophilin antibodies and either knock-out or IgG controls identified a total of 125 proteins. The probability of each protein being specifically associated with spinophilin in each sample was calculated, and proteins were ranked according to a chi(2) analysis of the probabilities from analyses of multiple samples. Spinophilin and the known associated proteins neurabin and multiple isoforms of protein phosphatase 1 were specifically detected. Multiple, novel, spinophilin-associated proteins ( myosin Va, calcium/calmodulin-dependent protein kinase II, neurofilament light polypeptide, postsynaptic density 95, alpha-actinin, and densin) were then shown to interact with GST fusion proteins containing fragments of spinophilin. Additional biochemical and transfected cell imaging studies showed that alpha-actinin and densin directly interact with residues 151-300 and 446-817, respectively, of spinophilin. Taken together, we have developed a multi-antibody, shotgun proteomics approach to characterize protein interactomes in native tissues, delineating the importance of knock-out tissue controls and providing novel insights into the nature and function of the spinophilin interactome in mature striatum. Molecular & Cellular Proteomics 9:1243-1259, 2010.