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

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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.