Mapping sites of aspirin-induced acetylations in live cells by quantitative acid-cleavable activity-based protein profiling (QA-ABPP).
Mapping sites of aspirin-induced acetylations in live cells by quantitative acid-cleavable activity-based protein profiling (QA-ABPP).
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DOI：
10.1038/srep07896
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发表时间：
2015-01-20
影响因子：
4.6
通讯作者：
Lin Q
中科院分区：
文献类型：
--
作者：
Wang J;Zhang CJ;Zhang J;He Y;Lee YM;Chen S;Lim TK;Ng S;Shen HM;Lin Q
小分子探针的发展及其在生物学和药物开发中的应用，对靶标的识别和作用机制的理解是一个挑战。例如，尽管阿司匹林已经被广泛使用了100多年，但它的分子靶标还没有得到充分的表征。为了应对这一挑战，我们开发了一种新的技术，称为基于酸可切割活性的定量蛋白质图谱，它结合了以下两个部分：(I)基于活性的蛋白质图谱和iTRAQ™定量蛋白质组学，用于鉴定目标蛋白质；(Ii)基于酸可裂解连接子的蛋白质图谱，用于鉴定具有特定结合位点的多肽。众所周知，阿司匹林与其靶蛋白的反应会导致乙酰化。因此，我们使用基于阿司匹林的探针将上述技术应用于人类癌症HCT116细胞。我们鉴定了1110个目的蛋白和2775个具有准确乙酰化位点的多肽。通过将这两组数据关联起来，523种蛋白质被确定为阿司匹林的靶标。我们使用不同的生物学方法来验证阿司匹林在抑制蛋白质合成和诱导自噬方面的作用，这些作用是从阿司匹林靶标图谱的通路分析中得出的。该技术广泛应用于药物发现和生物学领域的靶标识别，尤其适用于共价药物的识别。
Target-identification and understanding of mechanism-of-action (MOA) are challenging for development of small-molecule probes and their application in biology and drug discovery. For example, although aspirin has been widely used for more than 100 years, its molecular targets have not been fully characterized. To cope with this challenge, we developed a novel technique called quantitative acid-cleavable activity-based protein profiling (QA-ABPP) with combination of the following two parts: (i) activity-based protein profiling (ABPP) and iTRAQ™ quantitative proteomics for identification of target proteins and (ii) acid-cleavable linker-based ABPP for identification of peptides with specific binding sites. It is known that reaction of aspirin with its target proteins leads to acetylation. We thus applied the above technique using aspirin-based probes in human cancer HCT116 cells. We identified 1110 target proteins and 2775 peptides with exact acetylation sites. By correlating these two sets of data, 523 proteins were identified as targets of aspirin. We used various biological assays to validate the effects of aspirin on inhibition of protein synthesis and induction of autophagy which were elicited from the pathway analysis of Aspirin target profile. This technique is widely applicable for target identification in the field of drug discovery and biology, especially for the covalent drugs.