Global, site-specific analysis of neuronal protein S-acylation.
Global, site-specific analysis of neuronal protein S-acylation.
复制标题
DOI：
10.1038/s41598-017-04580-1
复制
发表时间：
2017-07-05
影响因子：
4.6
通讯作者：
Choudhary JS
中科院分区：
文献类型：
--
作者：
Collins MO;Woodley KT;Choudhary JS
蛋白质S-酰化（棕榈酰化）是一种可逆的脂质修饰，是动态膜-蛋白质相互作用的重要调节剂。蛋白质组学方法已经发现了许多假定的棕榈酰化蛋白质，然而，缺乏全面的棕榈酰化位点表征的方法。我们证明了一个定量的位点特异性酰基生物素交换（ssABE）的方法，允许识别906个假定的棕榈酰化位点641蛋白从小鼠前脑。62%的位点映射到已知的棕榈酰化蛋白质，并且从文献中已知102个单独的棕榈酰化位点。54%的棕榈酰化位点映射到突触蛋白，包括许多GPCR、受体/离子通道和外周膜蛋白。在棕榈酰化的肽的子集上也鉴定了磷酸化位点，这首次证明了通过质谱法共同鉴定这些修饰。棕榈酰化位点在超过一半的棕榈酰酰基转移酶（PAT）家族上被鉴定，所述家族介导蛋白质棕榈酰化，包括活性位点硫酯连接的棕榈酰中间体。不同的棕榈酰化基序和网站拓扑结构被确定为完整的膜和可溶性蛋白，表明相关的PAT特异性和棕榈酰化功能的潜在差异。ssABE允许棕榈酰化位点的全局识别以及PAT的活性位点修饰状态的测量，使得能够在系统水平上研究棕榈酰化。
Protein S-acylation (palmitoylation) is a reversible lipid modification that is an important regulator of dynamic membrane-protein interactions. Proteomic approaches have uncovered many putative palmitoylated proteins however, methods for comprehensive palmitoylation site characterization are lacking. We demonstrate a quantitative site-specific-Acyl-Biotin-Exchange (ssABE) method that allowed the identification of 906 putative palmitoylation sites on 641 proteins from mouse forebrain. 62% of sites map to known palmitoylated proteins and 102 individual palmitoylation sites are known from the literature. 54% of palmitoylation sites map to synaptic proteins including many GPCRs, receptors/ion channels and peripheral membrane proteins. Phosphorylation sites were also identified on a subset of peptides that were palmitoylated, demonstrating for the first time co-identification of these modifications by mass spectrometry. Palmitoylation sites were identified on over half of the family of palmitoyl-acyltransferases (PATs) that mediate protein palmitoylation, including active site thioester-linked palmitoyl intermediates. Distinct palmitoylation motifs and site topology were identified for integral membrane and soluble proteins, indicating potential differences in associated PAT specificity and palmitoylation function. ssABE allows the global identification of palmitoylation sites as well as measurement of the active site modification state of PATs, enabling palmitoylation to be studied at a systems level.