Quantitative analysis of protein phosphorylation on a system-wide scale by mass spectrometry-based proteomics.

Systems biology at the molecular level is concerned with networks of interacting molecules, their structure, and dynamic response to perturbations that give rise to systems' properties that determine measurable, macroscopic phenotypes. At any time, in any cell, multiple types of molecular networks are concurrently active. One of the most important known regulatory systems in eukaryotic cells is reversible protein phosphorylation catalyzed by protein kinases and phosphatases, respectively. Therefore, it is essential to understand and eventually model the protein phosphorylation-mediated informational fluxes in cells from sensors and signaling systems to effector molecules, to comprehensively analyze the dynamic system of kinases/phosphatases and their substrates and to determine the basic rules of information processing in cells. In this chapter, we describe the protocols necessary to comprehensively and quantitatively measure the phosphorylation-modulated informational networks in cells. The pipeline relies on the selective, quantitative isolation of phosphopeptides generated by the tryptic digestion of complex protein mixtures and their subsequent mass spectrometric and computational analysis. We believe that the protocols and data processing tools described in this chapter will be a valuable resource for biologists interested in the analysis of protein phosphorylation-based signal transduction.