Quantitative analysis of synaptic phosphorylation and protein expression
Quantitative analysis of synaptic phosphorylation and protein expression
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DOI：
10.1074/mcp.m700170-mcp200
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发表时间：
2008-04-01
影响因子：
7
通讯作者：
Burlingame, Alma L.
中科院分区：
文献类型：
--
作者：
Trinidad, Jonathan C.;Thalhammer, Agnes;Burlingame, Alma L.
突触后密度(PSD)信号机制包含具有多种功能的蛋白质。PSD成分的脑区特异性变化介导了对突触激活的不同生理反应。我们已经开发了基于质谱学的方法来综合比较突触后密度的生化准备中存在的蛋白质的相对蛋白质表达和磷酸化状态。使用这些方法，我们测定了2159个蛋白质和1564个磷酸化位点在小鼠大脑皮质、中脑、小脑和海马区的PSD样品中的相对表达。这些实验使用独立的生物复制进行了两次，这使我们能够评估该系统中的实验和生物变异性。在蛋白质表达方面，聚类分析表明，已知的功能相关蛋白质显示出协调的突触表达。因此，被鉴定为与已知蛋白质复合体共同聚集的蛋白质是以前未被识别的成分的主要候选者。在磷酸化程度方面，我们在N-甲基-D-天冬氨酸受体上观察到比alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic酸(AMPA)受体更广泛的磷酸化位点，这与N-甲基-D-天冬氨酸受体在处理突触传递模式中的中心作用一致。海马区的平均激酶和磷酸酶水平最高，这与该脑区存在较高的总磷酸肽丰度相关。这些发现表明，在改变突触强度时，海马体比其他脑区更大程度地利用可逆的蛋白质磷酸化。
The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry-based methods to comprehensively compare both relative protein expression and phosphorylation status from proteins present in biochemical preparations of postsynaptic density. Using these methods, we determined the relative expression of 2159 proteins and 1564 phosphorylation sites in PSD preparations from murine cortex, midbrain, cerebellum, and hippocampus. These experiments were conducted twice using independent biological replicates, which allowed us to assess the experimental and biological variability in this system. Concerning protein expression, cluster analysis revealed that known functionally associated proteins display coordinated synaptic expression. Therefore, proteins identified as co-clustering with known protein complexes are prime candidates for assignment as previously unrecognized components. Concerning degree of phosphorylation, we observed more extensive phosphorylation sites on N-methyl-D-aspartate (NMDA) receptors than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, consistent with the central role of N-methyl-D-aspartate receptors in processing synaptic transmission patterns. Average kinase and phosphatase levels were highest in the hippocampus, correlating with a higher overall phosphopeptide abundance present in this brain region. These findings suggest that the hippocampus utilizes reversible protein phosphorylation to a greater extent than other brain regions when modifying synaptic strength.