A scalable proteogenomic framework for dissecting phospho-signaling pathways in primary immune cells

Signaling networks modulated by post-translational modifications orchestrate cellular responses to external cues. Traditional approaches to study these pathways lack the throughput to systematically capture the causal architecture of these signaling pathways at scale. Here, we present an integrated proteogenomic framework that combines saturating genetic perturbations with high-throughput proteomics to systematically map cytokine-induced signaling in primary human T cells. Supporting this framework is simplePhos, a streamlined, low-input phosphoproteomics workflow that enables scalable, time-resolved analysis without the requirement for specialized equipment or robotics. We extensively validate the simplePhos pipeline by applying inflammatory stimuli, including type I and II interferons, lipopolysaccharide, and Sendai virus to primary T cells and myeloid cells, establishing foundational datasets in these treatment contexts. Ultimately, using type I interferon signaling in genetically modified T cells as a model, we demonstrate that combined application of genetic alterations and proteomic analyses can map key signaling nodes in primary immune cells. This represents a powerful strategy to mechanistically interrogate phospho-signaling networks in human immune cells, with broad applications in translational immunology and therapeutic development.