Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis.
Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis.
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DOI：
10.1021/pr300536k
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发表时间：
2012-09-07
影响因子：
4.4
通讯作者：
Peng J
中科院分区：
文献类型：
--
作者：
Na CH;Jones DR;Yang Y;Wang X;Xu Y;Peng J
蛋白泛素化是调节神经发育、突触可塑性、学习记忆等重要的翻译后修饰，其失调与神经系统疾病的发病有关。本文报道了一种新开发的单克隆抗体对大鼠脑中泛素化蛋白质组（ubiquitome）的系统分析，该抗体可识别胰蛋白酶化肽（K-GG肽）中赖氨酸残基上的二甘氨酸标签。初步抗体特异性分析表明，该抗体可区分K-GG肽与线性GG肽或由碘乙酰胺衍生的伪K-GG肽。为了评估在数据库搜索中K-GG肽匹配的错误发现率，我们引入了一个无效实验，使用不含此类肽的细菌裂解液。然后用强阳离子交换（SCX）预分离或不使用该抗体富集分析脑泛素组。在SCX层析中，虽然绝大多数K-GG肽在含有至少三个正电荷肽的组分中被检测到，但具有两个正电荷的特定K-GG肽（例如蛋白质n端乙酰化和c端非k /R肽）也在早期组分中被鉴定出来。c端K-GG肽的可靠性也得到了广泛的研究。最后，我们收集了921种蛋白质中2064个肽的1786个K-GG位点的数据集，并通过光谱计数估计了它们的丰度。该研究揭示了突触前区域关键成分（如Bassoon， NSF, SNAP25, synapsin, synaptotagmin, syntaxin）和突触后密度（如PSD-95， GKAP, CaMKII，以及NMDA， AMPA， GABA， 5 -羟色胺和乙酰胆碱受体）的广泛泛素化事件。我们还确定了淀粉样蛋白前体蛋白和α突触核蛋白上的泛素化位点，它们分别被认为是阿尔茨海默病和帕金森病的病原体。由于K-GG肽也可以由Nedd8或ISG15修饰的蛋白质产生，我们对大脑中的这些蛋白质进行了量化，发现它们的水平低于泛素的2%。总之，本研究证明了大量神经元蛋白被泛素化修饰，并为分析大脑中的泛素组提供了一种可行的方法。
Protein ubiquitination is an essential posttranslational modification regulating neurodevelopment, synaptic plasticity, learning and memory, and its dysregulation contributes to the pathogenesis of neurological diseases. Here we report a systematic analysis of ubiquitinated proteome (ubiquitome) in rat brain using a newly developed monoclonal antibody that recognizes the diglycine tag on lysine residues in trypsinized peptides (K-GG peptides). Initial antibody specificity analysis showed that the antibody can distinguish K-GG peptides from linear GG peptides or pseudo K-GG peptides derived from iodoacetamide. To evaluate the false discovery rate of K-GG peptide matches during database search, we introduced a null experiment using bacterial lysate that contains no such peptides. The brain ubiquitome was then analyzed by this antibody enrichment with or without strong cation exchange (SCX) prefractionation. During SCX chromatography, although the vast majority of K-GG peptides were detected in the fractions containing at least three positive charged peptides, specific K-GG peptides with two positive charges (e.g. protein N-terminal acetylated and C-terminal non-K/R peptides) were also identified in early fractions. The reliability of C-terminal K-GG peptides was also extensively investigated. Finally, we collected a dataset of 1786 K-GG sites on 2064 peptides in 921 proteins and estimated their abundance by spectral counting. The study reveals a wide range of ubiquitination events on key components in presynaptic region (e.g. Bassoon, NSF, SNAP25, synapsin, synaptotagmin, and syntaxin) and postsynaptic density (e.g. PSD-95, GKAP, CaMKII, as well as receptors for NMDA, AMPA, GABA, serotonin, and acetylcholine). We also determined ubiquitination sites on amyloid precursor protein and alpha synuclein that are thought to be causative agents in Alzhermer’s and Parkinson’s disorders, respectively. As K-GG peptides can also be produced from Nedd8 or ISG15 modified proteins, we quantified these proteins in the brain and found that their levels are less than 2% of ubiquitin. Together, this study demonstrates that a large number of neuronal proteins are modified by ubiquitination, and provides a feasible method for profiling the ubiquitome in the brain.
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影响因子：
3.7
作者：
Li W;Bengtson MH;Ulbrich A;Matsuda A;Reddy VA;Orth A;Chanda SK;Batalov S;Joazeiro CA
通讯作者：
Joazeiro CA
影响因子：
4.8
作者：
Dammer, Eric B.;Na, Chan Hyun;Peng, Junmin
通讯作者：
Peng, Junmin
影响因子：
7.4
作者：
Liu, HB;Sadygov, RG;Yates, JR
通讯作者：
Yates, JR
影响因子：
64.5
作者：
Krueger, Marcus;Moser, Markus;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
64.8
作者：
Hochstrasser, Mark
通讯作者：
Hochstrasser, Mark