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

Area of Science:

• Immunology
• Proteomics
• Systems Biology

Background:

• Cellular responses rely on complex signaling networks modulated by post-translational modifications.
• Existing methods for studying these pathways lack the scale and throughput for systematic causal architecture mapping.
• Understanding cytokine-induced signaling is crucial for immunology and therapeutic development.

Purpose of the Study:

• To present an integrated proteogenomic framework for systematically mapping cytokine-induced signaling pathways.
• To introduce simplePhos, a streamlined phosphoproteomics workflow for scalable, time-resolved analysis.
• To demonstrate a powerful strategy for interrogating phospho-signaling networks in human immune cells.

Main Methods:

• Developed an integrated proteogenomic framework combining genetic perturbations and high-throughput proteomics.
• Utilized simplePhos, a low-input phosphoproteomics workflow for scalable analysis.
• Applied the framework and workflow to primary human T cells and myeloid cells under various inflammatory stimuli.

Main Results:

• Established foundational proteogenomic datasets for cytokine-induced signaling in primary immune cells.
• Validated the simplePhos pipeline across multiple inflammatory stimuli (interferons, LPS, Sendai virus).
• Successfully mapped key signaling nodes in type I interferon signaling using the integrated framework.

Conclusions:

• The integrated proteogenomic framework and simplePhos workflow enable systematic mapping of signaling pathways in primary immune cells.
• This approach provides a powerful strategy for mechanistic interrogation of phospho-signaling networks.
• The findings have broad applications in translational immunology and the development of novel therapeutics.