Collaborative Research: MFB: Deciphering the Logic of PTM Crosstalk via Novel Chemical Technology: Histones and Beyond

在化学系发起的生物技术分子基础征集活动的支持下，与分子和细胞生物科学系的遗传机制和系统以及合成生物学集群合作，哈佛大学的菲利普科尔和圣路易斯华盛顿大学的本杰明加西亚将开发一种新的方法来破译翻译后修饰（PTM）在细胞蛋白质中相互影响。在过去的三十年里，使用质谱法和相关方法鉴定细胞蛋白质修饰方面出现了爆炸性增长，从而产生了令人难以置信的分子多样性。在这些情况中，只有少数情况下，人们才能理解这些修饰（通常以簇的形式发生）如何在同一蛋白质分子中相互影响。在这项合作研究努力下开发的实验程序将结合联合收割机设计的酶，小分子和质谱的力量，以阐明这些蛋白质修饰簇的语言和意义，这将告知基因和途径如何调节以及哺乳动物细胞如何发育。 这项研究将允许研究生和博士后研究员获得蛋白质化学和质谱学的专门培训。用于表征蛋白质修饰的新方法和试剂将与广大研究人员共享，以推动实验可行性的极限。该项目还包括一个推广计划，以加强对STEM感兴趣的大学和高中学生的教育。 这项合作研究项目是在NSF首届生物技术分子基础倡议下进行的，利用了分选酶的设计和部署能力，用于生物技术目的，采用最先进的质谱法。这两种不同的技术被用来开始解决生物学和生物技术中的一个重大挑战;即，我们如何全面和完整地分析表达的蛋白质组，并以一种可能定制的方式来最终个性化的方法。 这里采取的科学方法集中在开发一种新的化学酶策略，标记CUT和PASTE，从细胞蛋白质中分离大的纯化片段，以定量表征翻译后修饰（PTM）及其生物输出之间的相互作用。科尔/加西亚团队的策略利用了工程化分选酶沿着短的合成设计师化学标签的使用，这些标签将驱动同位素标记和亲和或荧光部分的掺入，以使用串联质谱和计算分析有效和定量地确定组蛋白和其他蛋白质的大的20至300个氨基酸肽段中的PTM。这项技术有希望成为变革性的，并有可能重新定义我们对蛋白质PTM逻辑的理解，驱动表观遗传机制和细胞网络和途径。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

With the support of the Molecular Foundations of Biotechnology Solicitation launched by the Division of Chemistry, in collaboration with the Genetic Mechanisms and Systems and Synthetic Biology Clusters in the Division of Molecular and Cellular Biosciences, Philip Cole of Harvard University and Benjamin Garcia of Washington University in St. Louis will develop a new method to decipher the logic of how post-translational modifications (PTMs) influence each other in cellular proteins. Over the past thirty years, there has been an explosion in the identification of modifications on cellular proteins using mass spectrometry and allied approaches giving rise to incredible molecular diversity. In but a few of these cases is it understood how these modifications, which often occur in clusters, influence each other within the same protein molecule. The experimental procedures to be developed under this collaborative research effort combine the power of designed enzymes, small molecules, and mass spectrometry to elucidate the language and meaning of these protein modification clusters that will inform how genes and pathways are regulated and how mammalian cells develop. This investigation will permit for graduate students and postdoctoral fellows to acquire specialized training in protein chemistry and mass spectrometry. The new methods and reagents for characterizing protein modifications will be shared with the broad community of researchers to push the limits of what is experimentally plausible. This project also includes an outreach program to enhance the education of college and high school students with an interest in STEM. This collaborative research project being conducted under the inaugural Molecular Foundations for Biotechnology initiative at NSF leverages the power of sortase enzyme design and deployment for biotechnological purposes with state-of-the-art methods in mass spectrometry. These two disparate techniques are being leveraged to begin to address a grand challenge in biology and biotechnology; namely, how do we comprehensively and completely analyze the expressed proteome and do so, in a manner that might be tailored to eventually individualize the approach. The scientific approach taken here is focused on developing a new chemoenzymatic strategy, labeled CUT and PASTE, to isolate large, purified fragments from cellular proteins for the purpose of quantitatively characterizing the interplay among post-translational modifications (PTMs) and their biological outputs. The Cole/Garcia team strategy exploits the use of engineered sortase enzymes along with short synthetic designer chemical tags that will drive incorporation of isotopic labels and affinity or fluorescent moieties to efficiently and quantitatively determine the PTMs in large, 20 to 300 amino acid peptide segments of histones and other proteins using tandem mass spectrometry and computational analysis. This technology has the promise to be transformative and to potentially redefine our understanding of the protein PTM logic that drives epigenetic mechanisms and cellular networks and pathways.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.