Exploring protein phosphorylation dynamics by mass spectrometry: pushing the boundaries of time resolved phosphoproteomics

Abstract

Here we used mass spectrometry to study protein phosphorylation across different experimental models: from single-embryos to patient-derived tissue samples. We combined different data acquisition strategies, from shotgun proteomics to get a global view of the phosphoproteome, to targeted measurement of specific phosphorylation sites of known biological relevance. Despite these differences, all the experimental chapters of this thesis share one feature: the measurement of protein phosphorylation levels throughout time. First, in chapter I we introduced basic concepts of mass spectrometry and how this technology prompted the rise of phosphoproteomics as a field of study. In chapter II, we focused on studying signal transduction in the MAPK-AKT-mTOR pathway by targeted phosphoproteomics. Using single reaction monitoring, we mapped the dynamic behavior of phosphorylation sites with known functionality after treating cells with different stimuli. We think this assay could be a valuable resource for those interested in reproducibly measuring phosphorylation dynamics of this protein network. In chapter III, we assessed the differences in specificity between the mitogen activated protein kinases ERK1 and ERK2, which could potentially explain why absence of ERK2 has been linked to more severe phenotypes on a variety of experimental models. On our most comprehensive chapter (IV), we explored changes in the phosphoproteome during the early cell divisions of X. laevis single-embryos, which showed our ability to distinguish cell cycle related phosphorylation in vivo. Next, our bioinformatic analysis of cell cycle phosphorylated substrates revealed enrichment of phosphorylation sites in highly disordered proteins. Using a model disordered protein (Ki-67), we showed that CDK1 driven phosphorylation modulates protein phase separation. Altogether, this allowed us to formulate a working hypothesis about how phosphorylation regulates protein condensation during cell cycle transitions, potentially explaining the drastic cellular reorganization observed in mitosis. In the last experimental chapter (V), we combined mass spectrometry technologies to assess changes in protein expression and protein phosphorylation in human intestinal tissue samples exposed to ischemia and reperfusion, in an attempt to understand the molecular mechanisms driving the pathophysiology of this clinical event. Finally, in chapter VI we share our conclusions and outlook for the field of phosphoproteomics and the study of protein phosphorylation.