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. 2023 Jan 6;14:94. doi: 10.1038/s41467-022-35740-1

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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. 5 — a Experimental scheme. MCF-7 cells were stimulated with TNF-α in the absence or presence of an I-kappa-B kinase inhibitor TPCA-1 (each condition in biological triplicate). For each replicate, DDA and DIA data were acquired on QE HF-X and timsTOF Pro instruments. The DIA data sets were processed by two software tools using either a project-specific DDA library or a DDA-independent library. b Number of TNF-α-regulated phosphosites from the analysis of HF-X data (left) and TIMS data (right) with different workflows. Phosphosites in response to TPCA-1 are those up-regulated by TNF-α and suppressed by TPCA-1 treatment, and their percentages over all up-regulated sites by TNF-α are indicated. c Enriched KEGG pathways based on the analysis of HF-X data (left) and TIMS data (right) with different workflows. Significantly enriched pathways (adjusted p < 0.05) are annotated in a color gradient. d Phophosites upregulated by TNF-α and included in the TNF-α pathway. They were identified in the analysis of HF-X data (left) and TIMS data (right) with different workflows. Significantly regulated sites (FC > 1.5, adjusted p < 0.05) are annotated in a color gradient. Results in b–d are based on phosphosite quantification data with missing value imputation. Source data are provided at 10.5281/zenodo.7409391.