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 Ti(4+)-IMAC phosphopeptide enrichment 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 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). PGE2 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 PGE2 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 PGE2 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.
From the ‡Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; §Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Erik L
EL
de Graaf
From the ‡Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; §Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Piero
P
Giansanti
From the ‡Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; §Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands m.altelaar@uu.nl a.j.r.heck@uu.nl.
A F Maarten
AF
Altelaar
From the ‡Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; §Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands m.altelaar@uu.nl a.j.r.heck@uu.nl.
Albert J R
AJ
Heck
D002846:Chromatography, Affinity / Q000379:methods*; D015232:Dinoprostone / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D014025:Titanium
D010748:Phosphopeptides; D014025:Titanium; D015232:Dinoprostone