New Sample Multiplexing Technologies to Identify Chemical Probes and Illuminate Ubiquitin Biology

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Summary Defects in ubiquitin (Ub) pathways are often responsible for cancer and devastating neurodegenerative diseases. Because of its central role in biological circuits and the potential for therapeutic intervention, the Ub system is an intense research area. Yet, Ub biology is highly complex, supported by over 500 Ub ligases and nearly 100 deubiquitinases. Currently, this complexity and our limited understanding of Ub biology players and their interactions are severe hindrances for targeted intervention in many diseases. Considering all possible ways to address this complexity, two important approaches have strong potential to fundamentally revolutionize our ability to unravel each protein's unique cellular role in Ub biology: i) sample multiplexing in mass spectrometry-based proteomics to improve throughput at proteome-wide depth, and ii) chemical proteomics to discover selective pharmacological tools to perturb and illuminate function. Through efforts within the previous grant cycle, the level of sample multiplexing was increased, allowing the proteome-wide comparison of expression and modification levels for 16 samples. In addition, a real-time database search doubled throughput while improving quantitative accuracy. In this proposal, we now turn our attention towards discovering chemical probes to study the Ub system through new chemical proteomics workflows where mass spectrometry is already a mainstay. In Aim 1, we will create two next-generation workflows—one for targeted and one for discovery proteomics—supporting isobaric tagging studies with 16plex reagents. The focus will be on fully integrating control over the scan decision process using external software and an API for protein quantification which will improve depth and quantitative precision. Given that reaction centers in most Ub system enzymes are cysteines (e.g., E2s, E3s, deubiquitinases), in Aim 2, we will modify the Aim 1 workflows to allow reactive cysteine profiling in chemical proteomics. Research under this aim will be completed with an eye toward establishing comprehensive protein-small molecule interaction landscapes for entire libraries of electrophilic compounds on a scale of thousands of molecules. Our goal in this Aim is to discover novel probes to Ub ligases and deubiquitinases. In Aim 3, we will create workflows to discover small molecules that induce Ub-mediated protein degradation. We will first modify both the discovery and targeted platforms from Aim 1 to allow us to work with starting amounts from cells growing in 96-well screening plates. Next, a plate-based cellular assay will be developed to measure the proteome-wide consequences of acute treatment with pools of compounds in each well, pinpointing compounds that directly engage the Ub-proteasome system to degrade their targets. The realization of these three aims will i) provide innovative new workflows for isobaric tagging studies, ii) generate new chemical probes as tools to study the diverse roles of Ub in cellular processes, and iii) create new precursors for therapeutic intervention.