Fig. 1: The optogenetic Akt1 system and experimental design of Optop-DIA.
From: An optogenetic-phosphoproteomic study reveals dynamic Akt1 signaling profiles in endothelial cells
a The optogenetic Akt1 system upon blue light activation. b Increased Akt1 phosphorylation after light illumination with co-expressed mCherry-CRY2-Akt1 and CIBN-GFP-CAAX constructs in HeLa CCL2 cells (Light intensity, 16 mW/cm2). c Live-cell imaging demonstrating that upon about 2 seconds blue light (488 nm laser) stimuli, Akt1 translocates to the plasma membrane. Green channel validating the CIBN-GFP-CAAX expression and ___location. d Light-induced Akt1 phosphorylation in optogenetic EA.hy 926 cells from 5 to 10 min and upon removal of light (light intensity, 2 mW/cm2). e Western blot of light intensity 0.01, 0.05, 0.1, 0.25, 0.5 to 1 mW/cm2 induced Akt1 phosphorylation in transduced EA.hy 926 cells. In (b), (d), and (e), the exogenous Akt1 and p-Akt1 signals are marked by dark red vertical line. f Systemic graph showing phosphoproteomic experimental design with light blue bars indicating light exposure and black bars for dark. Phosphopeptide enrichment and phosphoproteomics-DIA were performed on collected cell lysates, resulting in excellent consistency of sensitivity between different samples among Akt1 activation groups. The peptide fragment level information is used in DIA-MS for phosphosite (P-site) detection and quantification. Center bars denote mean of phosphopeptide enrichment efficiency percentage in the right panel. g Akt1 pT308 across all the samples and Akt1 substrates phosphorylation levels profiled by Optop-DIA. Left panel: The extracted ion chromatography (XIC graph) from DIA analysis for pT308 is used to infer the quantitative changes between samples. In the XIC, the peak groups above the middle line denote the MS2 level ion traces (i.e., peptide fragments) for the phosphopeptide signature, whereas the peak groups below the middle line denote the MS1 level ion traces (i.e., isotopic m/z of the intact phosphopeptide). Middle and right panels: The XIC graphs for the other four Akt1 substrates based on respective phosphopeptide signatures. The arrows indicate the identical Akt1 activation conditions as illustrated for Akt1 pT308 in the left panel. For (f, g), n = 2 biologically independent samples. For (b, d, e), two times of WB experiments were repeated independently with similar results. Source data are provided as a Source data file.
