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R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies
Mario Leutert1, Ricard A Rodríguez-Mias1, Noelle K Fukuda1
1Department of Genome Sciences, University of Washington, Seattle, WA, USA.
Molecular Systems Biology
|December 31, 2019
Summary
We developed rapid-robotic phosphoproteomics (R2-P2), an automated method for high-throughput phosphosite analysis. This technique enhances the study of complex cell signaling pathways, enabling deeper insights into cellular processes.
Area of Science:
- Proteomics
- Cellular Signaling
- Systems Biology
Background:
- Proteomics advancements allow identification of over 10,000 phosphosites.
- Current phosphoproteomics methods lack throughput, reproducibility, and robustness for complex signaling studies.
- Challenges include mapping kinase-substrate relationships, pathway crosstalks, and network inference.
Purpose of the Study:
- To introduce rapid-robotic phosphoproteomics (R2-P2), an automated, high-throughput method for phosphopeptide preparation.
- To overcome limitations in current phosphoproteomics analyses.
- To facilitate large-scale signaling studies with multiple perturbations.
Main Methods:
- Developed R2-P2, an end-to-end automated method using magnetic particles for phosphopeptide processing.
- Combined R2-P2 with data-independent acquisition mass spectrometry.
- Applied the method to study signaling dynamics in the yeast MAPK pathway.
Main Results:
- Demonstrated R2-P2 is rapid, robust, versatile, and high-throughput.
- Revealed broad and specific signaling events in yeast MAPK pathway branches (mating, high-osmolarity glycerol, invasive growth).
- Observed robust phosphorylation of downstream regulatory proteins and transcription factors.
Conclusions:
- R2-P2 significantly improves phosphoproteomics analysis.
- The method enables large-scale signaling studies involving hundreds of perturbations.
- Facilitates systems-level investigations into cellular signaling complexity.
