Proteomics approaches to decipher new signaling pathways

Cells have evolved intricate ways to propagate signals through signaling networks rich in crosstalks between signaling pathways and feedforward and feedback loops. The enzymatic products of phosphorylation-dependent signaling, namely phosphopeptides, can be identified and quantified systematically by mass spectrometry. Recent advances in the speed and sensitivity of mass spectrometers, combined with improvements in sample preparation and data analysis, are enabling the acquisition of increasingly large phosphopeptide datasets. Here, we discuss several considerations in experimental design that aid in unraveling direct and indirect phosphorylation events, as well as to identify crosstalks and feedback loops.