Exploring dynamic behavior with SmartPhos: an integrated pipeline for low-input, high-throughput Phosphoproteomics in clinical samples

Protein phosphorylation, a transient modification, plays a pivotal role in intracellular information processing. Changes in these processes significantly influence disease onset and development. Comprehensive studies examining changes in phosphorylation sites are crucial to unravel these complex, non-linear interrelations. However, a robust mass spectrometry-based phosphoproteomics workflow is challenging, given the low abundance of phosphorylated proteins in highly complex samples. In response to these challenges, we introduce SmartPhos, a cohesive multistep phosphoproteomics sample preparation and data analysis pipeline designed for longitudinal cell culture experiments and snapshot tissue samples. It reduces the need for starting material and aids in generating time-resolved data on phosphorylation sites. The user-friendly SmartPhos software streamlines data import, quality control, preprocessing, and exploratory analysis. To demonstrate its effectiveness, we used the SmartPhos pipeline to study the dynamics of growth factor-induced phosphorylation of the receptor tyrosine kinases EGFR and MET in non-small cell lung cancer (NSCLC) cells. In two days, we processed 156 samples, generating a report that included over 9,000 proteins and 40,000 class I phosphosites for each time point. The reliability and robustness of our pipeline were confirmed by comparing the phosphoproteome results with those from quantitative immunoblotting on the dynamics of selected phosphorylation sites. The SmartPhos pipeline enables a system-wide analysis to unravel dynamic cellular responses, while mathematical models facilitate the understanding of disease progression in individual patients. Together, they provide a holistic perspective on cellular information processing and yield critical insights for mathematical modeling to dissect disease-promoting mechanisms at a personalized level.