R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies

Recent developments in mass spectrometry-based proteomics have enabled systems-level studies of cellular signaling, where >10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges and fully exploit the potential of phosphoproteomics, we introduce rapid-robotic-phosphoproteomics (R2-P2), an end-to-end automated method that processes samples in a 96-well format, from a protein extract to mass spectrometry-ready phosphopeptides. R2-P2 uses magnetic particles for both protein sample cleanup and phosphopeptide enrichment. R2-P2 is more flexible, high-throughput, rapid, and robust than classical protocols. To showcase the method, we have applied it, in combination with data-independent acquisition mass spectrometry, to study signaling dynamics in the mitogen-activated protein kinase (MAPK) pathway in the yeast model Saccharomyces cerevisiae. Our results reveal broad and specific signaling events along the mating, the high-osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method greatly facilitates large-scale signaling studies involving hundreds of perturbations and will open the door to systems-level studies aiming to capture the complexity of signaling.