Low-Background Acyl-Biotinyl Exchange Largely Eliminates the Coisolation of Non-S-Acylated Proteins and Enables Deep S-Acylproteomic Analysis.
Low-Background Acyl-Biotinyl Exchange Largely Eliminates the Coisolation of Non-S-Acylated Proteins and Enables Deep S-Acylproteomic Analysis.
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低背景酰基-生物素交换很大程度上消除了非 S-酰基化蛋白质的共分离，并实现了深度 S-酰基蛋白质组分析。
DOI：
10.1021/acs.analchem.9b01520
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发表时间：
2019
影响因子：
7.4
通讯作者：
Yang,Wei
中科院分区：
文献类型：
--
作者：
Zhou,Bo;Wang,Yang;Yan,Yiwu;Mariscal,Javier;DiVizio,Dolores;Freeman,MichaelR;Yang,Wei
蛋白质s -酰化（也称为棕榈酰化）是一种常见的翻译后修饰，其失调在许多疾病的发病机制中起着关键作用。酰基-生物素基交换（ABE）是一种广泛用于s -酰基化蛋白富集的方法，具有捕获任何类型生物样品中整个酰基蛋白质组的潜力。在这里，我们发现目前的ABE方法由于非s -酰化蛋白的共分离而受到高背景的影响。背景可以通过在生物素- hpdp反应之前用2,2 ' -二硫代二吡啶堵塞剩余的游离半胱氨酸残基而大大降低。将低背景ABE （LB-ABE）方法与无标记蛋白质组学相结合，从人前列腺癌LNCaP细胞中鉴定出2 895个高置信度的酰基化蛋白（包括1 591个已知的酰基化蛋白），这是迄今为止在单一研究中鉴定出的最大的酰基蛋白质组数据集。免疫印迹分析证实了LNCaP细胞中5种已知和5种新的前列腺癌相关s -酰化蛋白的s -酰化，表明它们的s -酰化水平约为0.6-1.8%。总之，LB-ABE方法在很大程度上消除了非s -酰基化蛋白的共分离，并实现了深度s -酰基蛋白组学分析。与以前的ABE方法相比，该方法有望促进s -酰基蛋白质组的更全面和准确的定量。
ProteinS-acylation (also called palmitoylation) is a common post-translational modification whose deregulation plays a key role in the pathogenesis of many diseases. Acyl-biotinyl exchange (ABE), a widely used method for the enrichment ofS-acylated proteins, has the potential of capturing the entireS-acylproteome in any type of biological sample. Here, we showed that current ABE methods suffer from a high background arising from the coisolation of non-S-acylated proteins. The background can be substantially reduced by an additional blockage of residual free cysteine residues with 2,2′-dithiodipyridine prior to the biotin-HPDP reaction. Coupling the low-background ABE (LB-ABE) method with label-free proteomics, 2 895 high-confidence candidateS-acylated proteins (including 1 591 knownS-acylated proteins) were identified from human prostate cancer LNCaP cells, representing so-far the largestS-acylproteome data set identified in a single study. Immunoblotting analysis confirmed theS-acylation of five known and five novel prostate cancer-relatedS-acylated proteins in LNCaP cells and suggested that theirS-acylation levels were about 0.6–1.8%. In summary, the LB-ABE method largely eliminates the coisolation of non-S-acylated proteins and enables deepS-acylproteomic analysis. It is expected to facilitate a much more comprehensive and accurate quantification ofS-acylproteomes than previous ABE methods.