Effective Method for Accurate and Sensitive Quantitation of Rapid Changes of Newly Synthesized Proteins.
Effective Method for Accurate and Sensitive Quantitation of Rapid Changes of Newly Synthesized Proteins.
复制标题
对新合成蛋白质快速变化进行准确、灵敏定量的有效方法。
DOI：
10.1021/acs.analchem.0c01823
复制
发表时间：
2020
影响因子：
7.4
通讯作者：
Wu,Ronghu
中科院分区：
文献类型：
--
作者：
Tong,Ming;Suttapitugsakul,Suttipong;Wu,Ronghu
蛋白质合成在细胞中受到快速而严格的调节，以适应不断变化的细胞外和细胞内环境。快速蛋白质合成变化的精确定量可以提供对蛋白质功能和细胞活性的洞察，但由于缺乏有效的分析方法，实现这一目标非常具有挑战性。在这里，我们开发了一种有效的质谱为基础的方法命名为定量O-炔丙基嘌呤霉素标记（QOT）集成O-炔丙基嘌呤霉素标记，生物正交化学和多重蛋白质组学的快速蛋白质合成的全球和定量分析。目前的方法使我们能够准确地定量新合成的蛋白质的快速变化，因为与氨基酸及其类似物不同，tRNA可以立即被核糖体利用，而不需要被激活并与tRNA结合，因此不需要细胞饥饿或预处理。应用该方法定量测定了脂多糖（LPS）处理后THP-1巨噬细胞蛋白质合成的快速变化。对于15分钟标记，>3000个蛋白质被定量，并且238个蛋白质的合成被显著改变，包括转录因子和细胞因子。结果表明，蛋白质合成受到调节，以促进巨噬细胞响应LPS的蛋白质分泌。考虑到蛋白质合成的重要性，该方法可广泛应用于生物和生物医学研究领域中蛋白质合成的快速变化。
Protein synthesis is quickly and tightly regulated in cells to adapt to the ever-changing extracellular and intracellular environment. Accurate quantitation of rapid protein synthesis changes can provide insights into protein functions and cellular activities, but it is very challenging to achieve because of the lack of effective analysis methods. Here, we developed an effective mass spectrometry-based method named quantitative O-propargyl-puromycin tagging (QOT) by integrating O-propargyl-puromycin (OPP) labeling, bioorthogonal chemistry, and multiplexed proteomics for global and quantitative analysis of rapid protein synthesis. The current method enables us to accurately quantitate rapid changes of newly synthesized proteins because, unlike amino acids and their analogs, OPP can be utilized by the ribosome immediately without being activated and conjugated to tRNA, and thus cell starvation or pretreatment is not required. This method was applied to quantitate rapid changes of protein synthesis in THP-1 macrophages treated with lipopolysaccharide (LPS). For 15-min labeling, >3000 proteins were quantitated, and the synthesis of 238 proteins was significantly altered, including transcription factors and cytokines. The results demonstrated that protein synthesis was modulated to facilitate protein secretion in macrophages in response to LPS. Considering the importance of protein synthesis, this method can be extensively applied to investigate rapid changes of protein synthesis in the biological and biomedical research fields.