PO.ET02.07 · 实验与分子治疗
化学连接蛋白实现p53翻译后修饰的定量ELISA
Chemically ligated proteins enable quantitative ELISAs for p53 post-translational modifications
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言句：我们开发了化学连接的蛋白标准品，以实现针对p53关键翻译后修饰的完全定量ELISA，为癌症研究提供稳健的工具。
翻译后修饰(PTM)蛋白的分析通常依赖于质谱或蛋白质印迹等方法，这些方法非定量、耗时且成本高。ELISA提供了一种更快、更经济的替代方案；然而，大多数针对PTM的ELISA仅为半定量，因为它们缺乏用于生成标准曲线的参考标准品。为解决这一局限，我们开发了化学连接蛋白(CLP)，用作ELISA中的参考标准品，从而实现对PTM的准确检测和定量。该策略被应用于定量肿瘤抑制因子p53上的PTM，p53在50%的癌症肿瘤中发生突变或缺失。1 p53的PTM调控其在细胞中的活性，使得其精确测量对于理解p53功能至关重要。丝氨酸15和丝氨酸392处的磷酸化以及赖氨酸382处的乙酰化是与p53激活相关的充分表征的修饰。2 为支持针对该靶点的肿瘤学研究，我们开发了ELISA试剂盒来定量这些修饰。开发过程包括配对筛选以及生物学验证。使用经修饰、未修饰和非特异性修饰的p53的CLP，来筛选相对于p53未修饰位点或邻近修饰位点对各PTM位点特异的抗体对。生物学验证包括天然信号的检测、细胞提取物中的诱导以及磷酸酶处理后的信号降低。额外的生物学验证确认了所有样本类型的稀释线性、回收率和精密度。利用该策略，我们生成了三个能够准确定量p53 pS15、pS392和acK382的ELISA试剂盒，为推进p53调控与功能研究提供了稳健的工具。
1)Bailey, Matthew H等。"癌症驱动基因和突变的全面表征。"Cell第173卷,2期(2018)：371-385.e18。doi:10.1016/j.cell.2018.02.0602)Liu, Yanqing等。"p53修饰：强大守护者的精致装饰。"分子细胞生物学杂志第11卷,7期(2019)：564-577。doi:10.1093/jmcb/mjz060
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查看英文原文 English abstract
Introductory Sentence: We developed chemically ligated protein standards to enable fully quantitative ELISAs for key post-translational modifications of p53, providing robust tools for cancer research.
Analysis of post translationally modified (PTM) proteins often relies on methods such as mass spectrometry or western blotting, which are non-quantitative, time consuming and costly. ELISAs offer a faster and more economical alternative; however, most PTM-targeted ELISAs are only semi-quantitative because they lack reference standards to generate a standard curve. To address this limitation, we developed chemically ligated proteins (CLPs) that serve as reference standards in ELISAs, enabling accurate detection and quantification of PTMs. This strategy was applied to quantify PTMs on the tumor suppressor p53 which is mutated or deleted in 50% of cancer tumors. 1 PTMs of p53 regulate its activity in the cell, making their precise measurement critical for understanding p53 function. Phosphorylation at serine 15 and serine 392 and acetylation at lysine 382 are well-characterized modifications associated with p53 activation. 2 To support oncology studies of this target, we developed ELISA kits to quantify these modifications. Development involved pair screening as well as biological validation. CLPs of modified, unmodified, and non-specifically modified p53 were used to select antibody pairs specific to each PTM site over unmodified or proximal modification sites of p53. Biological validation included detection of native signal, induction in cell extracts, and signal reduction following phosphatase treatment. Additional biological validation confirmed linearity of dilution, recovery, and precision across all sample types. Using this strategy, we generated three ELISA kits capable of accurately quantifying p53 pS15, pS392, and acK382, providing robust tools to advance research on p53 regulation and function.
1)Bailey, Matthew H et al. “Comprehensive Characterization of Cancer Driver Genes and Mutations.” Cell vol. 173,2 (2018): 371-385.e18. doi:10.1016/j.cell.2018.02.0602)Liu, Yanqing et al. “p53 modifications: exquisite decorations of the powerful guardian.” Journal of molecular cell biology vol. 11,7 (2019): 564-577. doi:10.1093/jmcb/mjz060
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利益披露 Disclosure
T. Bailey, None.