Comprehensive identification of phosphorylation sites in postsynaptic density preparations
Comprehensive identification of phosphorylation sites in postsynaptic density preparations
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DOI：
10.1074/mcp.t500041-mcp200
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发表时间：
2006-05-01
影响因子：
7
通讯作者：
Burlingame, AL
中科院分区：
文献类型：
--
作者：
Trinidad, JC;Specht, CG;Burlingame, AL
在哺乳动物中枢神经系统中，称为突触后密度（PSD）的结构是一种致密的蛋白质复合物，其功能是检测和响应从突触前轴突末端释放的神经递质。这种分子机器中蛋白质磷酸化的调节对其成分的活性至关重要，这些成分包括神经递质受体、激酶/磷酸酶、支架分子和调节细胞骨架结构的蛋白质。为了表征 PSD 样品中蛋白质的磷酸化状态，我们将强阳离子交换 (SCX) 色谱法与 IMAC 结合起来。最初，通过阳离子交换分离胰蛋白酶肽，并通过反相色谱结合串联质谱进行分析，从而鉴定出大多数 SCX 级分中的磷酸肽。由于每个单独的级分都过于复杂，无法在单次 LC-MS/MS 运行中完全表征，因此我们通过对每个 SCX 级分执行 IMAC 来富集磷酸肽，相对于单独的任一方法（SCX 或 IMAC），所鉴定的磷酸肽至少增加了 3 倍。这使我们能够在突触后密度制剂中发现的所有蛋白质中，识别出 23%（1,264 种中的 287 种）至少有一个磷酸化位点。我们总共鉴定了 998 个独特的磷酸化肽，映射到 723 个独特的磷酸化位点。在所有磷酸化肽中，62%（998 个中的 621 个）至少确定了一个精确的磷酸化位点，与此类似，80% 的已鉴定磷酸化位点是新的。
In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and similar to 80% of identified phosphorylation sites are novel.