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

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 en-richment method. We demonstrate the applicability of this approach by monitoring the phosphoproteome dynamics of Jurkat T cells stimulated by prostaglandin E2 (PGE2) over six different time points, measuring in total 108 snap-shots 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). PGE2 is known to increase cellular cAMP levels, thereb