Time is of the essence for signaling molecules. Although enzyme kinetics suggest that some cell signaling events peak within seconds, most proteomics techniques cannot resolve events within 30 seconds of signal onset. Now Dengjel et al. report a proteomics technique that can measure protein phosphorylation with one-second resolution in a recent article in Nature Biotechnology.
Epidermal growth factor (EGF) receptors are receptor tyrosine kinases. Upon binding EGF, the receptors dimerize and autophosphorylate. The activated EGF receptor kinase then recruits and phosphorylates the signaling molecules Src homologous and collagen-like protein (Shc) and phospholipase Cg1.
To improve the time resolution of proteomic sampling, the authors needed to stop cellular signaling quickly. They quenched signaling events by snap-freezing reactions with ice cold ethanol. However, human reaction time is not fast enough to reliably start and stop reactions within seconds by hand. The authors designed a series of peristaltic pumps to collect cultured cells, initiate signaling events and quench reactions. The circuit of pumps ran continuously, with a constant flow rate. To quench reactions at 1, 5, 10 and 60 seconds after signal initiation (0 seconds), the authors varied the length of the tube transferring EGF-stimulated samples to the quenching chamber.
Stable isotope labeling by amino acids in culture (SILAC) allowed the authors to compare signaling events across time. SILAC uses different isotopes to label signaling products in different cultures. Resulting samples can then by analyzed together by mass spectrometry, so that the relative amount of each phosphopeptide can be determined at each time point. When they added EGF to each culture, the authors also added one of three arginine isotopes and one of three lysine isotopes.
Following EGF stimulation, the authors immunoprecipitated cell lysates with either an anti-EGF receptor or an anti-phosphotyrosine antibody. Then they digested immunoprecipitates with the serine protease trypsin and enriched samples for phosphorylated proteins with titanium dioxide. They analyzed processed samples with mass spectrometry.
Of the tyrosines in the EGF receptor sequence, tyrosines 1092 and 1197 were phosphorylated within 1 second and tyrosine 1172 was phosphorylated within 5 seconds of EGF stimulation. Phosphorylation of Shc occurred within 5 seconds of EGF stimulation, but phosphorylation of phospholipase Cγ1 peaked later, after 60 seconds of EGF stimulation.
In addition to clarifying the timecourse of cellular signaling, the authors suggest a practical application for their technique. Fast and slow signaling events might differentiate direct and indirect targets of drug action in high-throughput screens, according to the authors.