Single-step Enrichment by Ti4+-IMAC and Label-free Quantitation Enables In-depth Monitoring of Phosphorylation Dynamics with High Reproducibility and Temporal Resolution

Quantitative phosphoproteomics workflows traditionally involve additional sample labeling and fractionation steps for accurate and in-depth analysis. Here we report a high-throughput, straightforward, and comprehensive label-free phosphoproteomics approach using the highly selective, reproducible, and sensitive Ti4+-IMAC phosphopeptide enrichment method. We demonstrate the applicability of this approach by monitoring the phosphoproteome dynamics of Jurkat T cells stimulated by prostaglandin E-2 (PGE(2)) over six different time points, measuring in total 108 snapshots of the phosphoproteome. In total, we quantitatively monitored 12,799 unique phosphosites over all time points with very high quantitative reproducibility (average r > 0.9 over 100 measurements and a median cv < 0.2). PGE(2) is known to increase cellular cAMP levels, thereby activating PKA. The in-depth analysis revealed temporal regulation of a wide variety of phosphosites associated not only with PKA, but also with a variety of other classes of kinases. Following PGE(2) stimulation, several pathways became only transiently activated, revealing that in-depth dynamic profiling requires techniques with high temporal resolution. Moreover, the large publicly available dataset provides a valuable resource for downstream PGE(2) signaling dynamics in T cells, and cAMP-mediated signaling in particular. More generally, our method enables in-depth, quantitative, high-throughput phosphoproteome screening on any system, requiring very little sample, sample preparation, and analysis time.