Capturing the signalling dynamics of the MAPK-AKT-mTOR pathway in a single targeted phosphoproteomics assay

The MAPK-AKT-mTOR protein network integrates extra- and intracellular signals to determine cellular fate, regulating pivotal biological processes such as cell growth and metabolism. Due to this crucial role, pathway dysregulation has been implicated in multiple diseases, such as metabolic disorders and cancer. The MAPK-AKT-mTOR pathway consists of dozens of proteins and signal transduction is primarily driven by protein phosphorylation. Here, we present a targeted phosphoproteomics assay to study the phosphorylation dynamics of the MAPK-AKT-mTOR pathway in detail with high sensitivity and in a high throughput manner. By using a multi-protease approach, we increased the pathway coverage with phosphosites that were previously inaccessible. This novel approach yields the most comprehensive method for the detailed study of mTOR signalling to date (covering 150 phosphopeptides on more than 70 phosphoproteins), which can be applied to in vitro and in vivo systems and has the sensitivity to be compatible with small sample amounts. We demonstrate the feasibility of this assay to monitor the plasticity of MAPK-AKT-mTOR phosphorylation dynamics in response to cellular stimuli with high temporal resolution and amino acid residue specificity. We found highly dynamic phosphorylation events upon treatment with growth factors, revealing the sequential nature of phosphosites in this signalling pathway. Furthermore, starvation of glucose and amino acids showed upregulation of AKT-targets PRAS40T246 and FOXO3T32, highlighting the role of AKT in cellular response to starvation. These findings illustrate the potential of this assay to obtain new biological insight when monitoring dynamics of functional phosphosites. Highlights Robust targeted MS assay to study the phosphorylation dynamics of the MAPK-AKT-mTOR network Extended pathway coverage by application of multiple proteases for protein digestion Highly sensitive, high throughput and readily applicable assay for in vivo and in vitro systems Phosphorylation patterns of MAPK-AKT-mTOR network are highly dynamic and change upon stimulation with growth factors, amino acids and glucose Motivation The MAPK-AKT-mTOR protein network integrates extra- and intracellular signals to determine cellular fate, regulating pivotal biological processes such as cell growth and metabolism. Due to this crucial role, pathway dysregulation has been implicated in multiple diseases, such as metabolic disorders and cancer. Our understanding of the complex regulation of this intricate signalling network is incomplete and is hampered by the lack of analytical methods to study its phosphorylation dynamics in detail. In this study, we present a targeted phosphoproteomics assay to monitor the phosphorylation events of the MAPK-AKT-mTOR pathway with amino acid residue specificity and in a high throughput manner. We increased the pathway coverage with phosphosites that were previously inaccessible by the use of multiple proteases for protein digestion. This novel approach yields the most comprehensive method for the detailed study of MAPK-AKT-mTOR signalling to date, which can be applied to in vitro and in vivo human samples and has the sensitivity to be compatible with small amounts of starting material. Graphical abstract