Rapid and cell type specific nascent proteome labeling in the nervous system

Controlled protein synthesis is key to regulating gene expression and by shaping the varying proteomes of individual cell populations, plays an important role in the development of specialized cell functions. Despite advances in assessing cell type-specific transcriptomes, methods to interrogate the nascent proteome of individual cell populations have not kept pace. Here, we will develop and optimize an innovative and powerful method that we recently pioneered to achieve rapid labeling of newly synthesized proteins with cellular resolution. Our chemical-genetic method combines a puromycin-based labeling strategy involving a caged form of OP-puromycin (OPP) called PhAc-OPP with ectopic expression of the enzyme penicillin G acylase (PGA) in any cell population of interest. PhAc-OPP is unblocked and converted to OPP by PGA by enzymatic phenylacetyl blocking group removal, thus allowing OPP labeling of newly synthesized proteins in PGA- expressing cell populations. We have achieved several important preliminary milestones in demonstrating the efficacy of this method, including cell type-specific protein labeling in mouse primary neurons. To advance this method we call POPPi (PGA-dependent OPP incorporation) toward translational profiling of complex tissue cell populations in vivo, we have generated a PGA transgenic Drosophila strain that can be mated to any cell-type specific driver of choice. The use of Drosophila harnesses the extensive range of genetic drivers available for achieving cell type specific expression in the fly CNS with the rapid scalability of fly populations to facilitate profiling of rare cell populations. Preliminary testing supports our ability to label and capture neuronal and glial cell populations within isolated Drosophila brain explants using this approach. The first major goal of the current study is to quantitatively profile labeled nascent proteomes from discrete brain cell populations using TMT tagging and LC-MS/MS. This will enable us to determine whether nascent proteomes captured from targeted cell populations are truly cell type-specific and of sufficient depth to have utility in future biological applications of the method. A second goal of the study is to develop and optimize a dietary PhAc-OPP ingestion approach for proteome labeling. If successful, the ability to administer PhAc-OPP dietarily will elevate utility of the method by demonstrating its suitability for assessing the effects of physiological or pathological stimuli on translation that can only be applied to an intact fly. Collectively, this work forms key milestones in evaluating the potential for applying POPPi to quantitative profiling of the nascent proteome following physiological or pathological stimuli. Project Number: 1R21NS148839-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Ian Martin | Institution: OREGON HEALTH & SCIENCE UNIVERSITY, PORTLAND, OR | Award Amount: $429,000 | Activity Code: R21 | Study Section: Enabling Bioanalytical and Imaging Technologies Study Section[EBIT] View on NIH RePORTER: https://reporter.nih.gov/project-details/11351388