Chemically ligated proteins enable quantitative ELISAs for p53 post-translational modifications

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 AB0578_v1.0_111725