Technologies for PTM discovery and functional mapping p. 505

项目总结

Project Summary Post-translational modification (PTM) of proteins is a pervasive form of cell signaling that orchestrates numerous processes, including metabolism, cell mobility, cell cycle, and differentiation. As a result, improper regulation of PTMs is widely implicated in aberrant development and disease. Mechanistically, post-translational modification provides a rapid and largely reversible means to modulate protein activity and transduce signals. Thus, the proteome and its modifications represent a rich and informative experimental plane. Research that seeks to understand its dynamics will doubtless advance our understanding of fundamental biology and disease. Mass spectrometry (MS) is well-suited to proteomic analysis because it is highly sensitive, has the capacity to localize PTMs to a single amino acid, and, unlike antibody-based methods, does not require a priori knowledge of protein targets or post-translational modifications; indeed, MS has been used to catalog the complexity of various PTMs with great detail. That said, two major challenges remain. First, the field of proteomics almost uniformly relies on peptide cation analysis (i.e., positive electrospray). The consequence of this format is that acidic PTMs, and all PTMs contained in acidic regions of the proteome, are often difficult, or impossible, to detect because they do not ionize effectively under the standard low-pH conditions. We confront this problem by developing high-pH separations methodology along with negative electron transfer dissociation (NETD). NETD allows for the sequencing of peptide anions and should permit access to previously unobserved PTMs and portions of the proteome. The second major challenge is how to convert large PTM datasets to biological information. To counter this issue we propose a high-throughput technology, multiplexed assay for enzyme specificity (MAES), that converts discovery PTM data to functional information by en masse substrate-to-enzyme mapping. Mapping the enzymes responsible for a specific modification event is instructive because it places those modifications in the context of signaling molecules that direct biological function. Many of the DBPs in the Center will establish comprehensive lists of PTMs, which will serve as the foundation for further study. To provide direction for these studies, targeted, yet scalable, assays are necessary to map the enzymes that regulate modification sites of interest.