Fig 1.
DDA and DIA dual proteome profiling of Salmonella-infected epithelial cells.
(A) Venn diagram created using Venny (http://bioinfogp.cnb.csic.es/tools/venny/index.html) showing the total number of human (left Venn diagram) and S. Typhimurium (right) proteins identified using DDA (orange) and DIA (grey) data (both at FDR ≤ 0.01). Variability of DDA (B) and DIA (C) replicate samples represented in PCA plots. Green, cyan, blue, orange, purple and pink circles represent control (Ctrl), 2, 4, 8, 16 and 24 hpi samples, respectively.
Fig 2.
Heatmaps of significantly regulated (FDR ≤ 0.02) normalized human protein annotation enrichment scores across all six conditions (averaged expression values) assayed by data-independent acquisition (DIA).
Term enrichment was determined using the 1D annotation enrichment algorithm embedded in the Perseus software suite and p-values were corrected for multiple hypotheses testing using the Benjamini and Hochberg false discovery rate. Enriched annotation terms (FDR ≤ 0.02) in the categories (A) UniProt keywords, (B) GOBP (Biological Process), (C) general GO and (D) GOCC (Cellular Component) are shown as heatmaps and colour coded according to the 1D enrichment scores calculated [49], with red and green colouring indicating enriched and depleted annotations, respectively.
Fig 3.
Protein profile plots of significantly regulated (FDR ≤ 0.02) normalized human annotation enrichment scores across all 6 conditions (averaged expression values) assayed by data-independent acquisition (DIA).
Term enrichment was determined using the 1D annotation enrichment algorithm embedded in the Perseus software suite and p-values were corrected for multiple hypotheses testing using the Benjamini and Hochberg false discovery rate. Only corrected p-values ≤ 0.02 were considered. Selected GO terms and keyword annotations for with significantly altered protein abundancies were observed (i.e., extracellular matrix organization (GOBP, #26); upregulation at 24 hpi, cell adhesion (GOBP, #45); upregulation at 24 hpi, IL1-mediated signalling (GOBP, #27); upregulation at 2 hpi, integrin complex (keyword, #6); upregulation 24 hpi, actin cytoskeleton (GOCC, #26); downregulation 24 hpi, Ficolin-1-rich granule lumen (GOCC, #40); upregulation 2 hpi and downregulation 16 hpi, IFN-gamma signalling (GO, #8); upregulation 16 hpi, collagen binding (GO, #10); upregulation 24 hpi, proteasome complex (keyword, #22); down in control and upregulation 2 hpi) are shown for all setups analysed. Red and black asterisk above the profile plots means significant downregulation and upregulation of the corresponding proteins in the annotation group, respectively.
Fig 4.
Heatmaps of significantly regulated (FDR ≤ 0.05) normalized S. Typhimurium regulon enrichment scores and expression changes of their identified regulators across all infection conditions (averaged expression values) assayed by data-independent acquisition (DIA).
(A) Using a recently reported compilation of highly curated regulon gene targets identified by means of microarray or NGS-based gene expression studies [59], enrichment of specific regulons was determined using the 1D annotation enrichment algorithm embedded in the Perseus software suite. P-values were corrected for multiple hypotheses testing using the Benjamini and Hochberg false discovery rate. Enriched annotation terms (FDR ≤ 0.05) in the categories are shown as heatmaps and colour coded according to the 1D enrichment scores calculated [49], with red and green colouring indicating enriched and depleted annotations, respectively. ESP (early stationary growth phase), MEP (mid exponential phase), upregulated (UP) and downregulated (DN) genes as reported in [59]. (B) Heatmap representation of significantly regulated S. Tyhimurium regulators (FDR ≤ 0.01, corresponding normalized averaged z-scores) from the DIA analysis and corresponding to the regulated regulons shown in A. SsrB was also identified, but expression was not significantly regulated.
Fig 5.
Protein profile plots of significantly regulated (FDR ≤ 0.05) normalized S. Typhimurium annotation enrichment scores across all 5 infection conditions (averaged expression values) assayed by data-independent acquisition (DIA).
Term enrichment was determined using the 1D annotation enrichment algorithm embedded in the Perseus software suite and p-values were corrected for multiple hypotheses testing using the Benjamini and Hochberg false discovery rate. Only corrected p-values ≤ 0.05 were considered. Selected GO terms and keyword annotations for with significantly altered protein abundancies were observed (i.e., SPI-1 (#4); downregulation at 16 and 24 hpi, histidine biosynthesis (keyword, #8); upregulation at 16 and 24 hpi) are shown for the 5 infection setups analysed. A black asterisk above the profile plots means significant upregulation (by 1D annotation enrichment of the corresponding proteins in the annotation group. Besides, some other categories (SPI-2 (#4), chemotaxis (GO, #4), structural constituent of the ribosome (GOMF, #14) and cell division (GOBP, #9) of which the corresponding protein members were all found to be significantly regulated based on t-testing are shown as profile plots.