Post-translational phenotypic profiling through nucleotide barcode sequencing

摘要 DNA合成和测序的进步现在使大规模实验成为可能， 系统地探索序列变异如何影响分子和细胞功能。 转录调控已被证明特别适合大规模平行实验 这些研究揭示了序列变异如何影响启动子和增强子的活性， 以及转录因子。这些实验依赖于高通量测序， 在转录过程中产生的mRNA作为调节活性的直接读出。这 优雅的方法不能直接扩展到研究其他重要的生物模式， 然而，监管。 翻译后蛋白质修饰是几乎所有细胞信号通路的基础。尽管 重要的是，我们缺乏一般的方法来评估序列变异如何影响后， 翻译调节我们将通过一项广泛适用的战略来解决这一差距， 蛋白质修饰，如磷酸化或调节性降解， 测序读数。在这里，我们建议开发这种技术，并验证它， 证明了蛋白质水平的调节是如何受到靶蛋白变异的影响， 修饰酶，以及细胞更广泛的遗传背景。

ABSTRACT Advances in DNA synthesis and sequencing now enable large-scale experiments that systematically explore how sequence variation affects molecular and cellular function. Transcriptional regulation has proven particularly amenable to massively parallel experiments that have revealed how sequence variation affects the activities of promoters and enhancers as well as transcription factors. These experiments have relied on high-throughput sequencing of the mRNAs produced during transcription as a direct read-out of regulatory activity. This elegant approach cannot be directly extended to study other important modes of biological regulation, however. Post-translational protein modification underlies nearly all cell signaling pathways. Despite its importance, we lack general approaches to assess how sequence variation affects post- translational regulation. We will address this gap with a broadly applicable strategy to link protein modifications, such as phosphorylation or regulated degradation, with a deep sequencing readout. Here we propose to develop this technique and validate it by demonstrating how protein-level regulation is affected by variation in the target protein, the modifying enzyme, and the broader genetic context of the cell.