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. 2023 Jun 16;14:3599. doi: 10.1038/s41467-023-39347-y

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Experimental design to study EGF time course stimulation in the presence of three inhibitors of downstream pathways: EGFRi (lapatinib), PI3Ki (wortmannin) and MEKi (PD0325901). Each condition was performed in quadruplicates. b Significant proteins after 10 min of EGF stimulation (two-sided, two-sample t-test, log2 FC (10 min vs control) >1.5, q value < 0.01, n = 4 biological replicates) that are not upregulated after EGFR inhibition. MEK dependent are those regulated after 10 min of EGF stimulation that do not show upregulation after PD0325901 treatment. PI3K dependent are those regulated after 10 min of EGF stimulation that do not show upregulation after wortmannin treatment. Gray indicates that the site was found to be dependent on the indicated kinase. Last column show the Pearson correlation between the profile of those sites in hybrid-DIA and SureQuant. c Profile plot of representative regulated sites by EGF (two-sided, two-sample t-test, log2 FC (10 min vs control) >1.5, q-value < 0.01). Dots indicate the absolute ratio Endogenous to Heavy standard. In blue, data from hybrid-DIA quantification (n = 4 biological replicates); and in pink, data from SureQuant quantification (n = 4 biological replicates). Line indicate the average of the experimental replicates, and the error bars the standard error of the mean. d Summary of results obtained from the extracted DIA scans from the hybrid-DIA experiment. e Kinase activity inference analysis obtained from RoKAI using the discovery analysis data from the hybrid-DIA experiment. Color of the dots reflect the value of the inferred kinase activity calculated by ROKAI21 (higher values correlate with higher kinase activity in the sample). Size of the dots is proportional to the -log10 of the FDR corrected p-value. Asterisks indicate FDR q value < 0.01. Source data are provided as a Source Data file.