Despite the wealth of computational techniques designed to predict responses from RTK signaling, the data available for these efforts is relatively poor. Major shortcomings include lack of site specificity, relative quantification, and poor temporal resolution. We have developed new mass spectrometry-based methods that improve measurements of signaling dynamics. MARQUIS is a targeted measurement technique that uses synthetic standard peptides to report absolute amounts of select peptides (copies/cell), a measurement that is important for mechanistic models and allows comparison between distinct phosphorylation sites. Additionally, we have generated methods to study signaling dynamics with high temporal (10s) resolution, and are investigating the immediate-early signaling dynamics following RTK stimulation. This method has uncovered network-wide phosphorylation changes as early as 10s, which could hold the key to explaining nearly immediate phenotypic responses observed with live-cell imaging.
