Chemical Approaches to Characterizing Protein Post-translational Modifications by Mass Spectrometry

摘要 细胞中的大多数生物事件在一定程度上是由蛋白质翻译后修饰介导的。为 例如，由激酶酶催化的异常蛋白磷酸化与多种不同的 癌症。类似地，组蛋白脱乙酰酶控制的组蛋白未受调控的乙酰化状态 (HDAC)蛋白，可导致转录的表观遗传变化，最终导致疾病。关键是 健康和疾病状态的特征是对蛋白质作用的详细的分子理解 翻译后修饰，如磷酸化和乙酰化，对蛋白质功能和 互动。重要的是，调节翻译后修饰的酶，包括激酶和HDAC 蛋白质是药物治疗的靶点。然而，将蛋白质修饰与下游生物联系起来的工具 活动往往有限或无法获得，这阻碍了疾病特征和药物方面的进展。 发展。 NIGMS资助的Pflum实验室项目满足了开发创新化学品的迫切需要 在细胞生物学中发现蛋白质修饰酶未预料到的功能的方法。在工作中 通过蛋白质磷酸化，我们在过去10年中率先将ATP类似物应用于激酶-- 催化标记反应开发一套检测激酶-底物对和多蛋白质的方法 细胞中的复合体。此补充资金申请将通过应用我们的创新技术来支持此项目 与生物学家合作，提供解决复杂生物学问题的工具。在我们对蛋白质乙酰化的研究中，我们 在过去的几年里已经证明了使用诱捕突变体来发现非组蛋白的力量 HDAC1的底物，这揭示了HDAC1蛋白在细胞生物学中意想不到的作用。在这 补充资金申请，我们将应用这一强大的诱捕策略来发现 HDAC1在镰状细胞贫血模型系统中的作用，这将揭示镰状细胞贫血中未知的分子机制 细胞和可能的新疗法。重要的是，这些研究将提供一个理想的培训场地来揭示 以蛋白质组学为基础的质谱学、生物化学和细胞生物学的博士前候选人 为进入生物医学大军做好准备。

Summary Most biological events in the cell are mediated at some level by protein post-translational modifications. For example, aberrant protein phosphorylation catalyzed by kinase enzymes is linked to a wide variety of cancers. Similarly, the unregulated acetylation state of histone proteins, controlled by histone deacetylase (HDAC) proteins, can lead to epigenetic changes in transcription and ultimately disease. Key to characterizing both healthy and disease states is a detailed molecular understanding of the role of protein post-translational modifications, such as phosphorylation and acetylation, on protein function and interactions. Importantly, enzymes regulating post-translational modifications, including kinase and HDAC proteins, are targets of drug treatment. Yet, tools linking protein modifications to downstream biological activities are often limited or unavailable, which has stalled progress in disease characterization and drug development. The NIGMS-funded projects in the Pflum lab address the critical need to develop innovative chemical approaches to discover unanticipated functions of protein modifying enzymes in cell biology. In our work with protein phosphorylation, we have pioneered in the last 10 years application of ATP analogs in kinase- catalyzed labeling reactions to develop a suite of methods to probe kinase-substrate pairs and multi-protein complexes in cells. This supplemental funding application will support this project by applying our innovative tools to complex biological problems in collaboration with biologists. In our work with protein acetylation, we have demonstrated in the last several years the power of using trapping mutants to discover non-histone substrates of HDAC1, which has revealed unexpected roles of HDAC1 proteins in cell biology. In this supplemental funding application, we will apply this powerful trapping strategy to discover the substrates of HDAC1 in sickle cell anemia model systems, which will reveal unanticipated molecular mechanisms in sickle cell and possible new therapeutics. Importantly, these studies will provide an ideal training ground to expose the pre-doctoral candidate to proteomics-based mass spectrometry, biochemistry, and cell biology to prepare for entry into the biomedical workforce.