![Phosphoproteomics analysis suggests modulation of cAMP/Ca2+ signalling pathways in pig sinus node upon GLP-1 infusion. (A) Protocol of infusion of GLP-1 or vehicle prior to tissue collection (n = 4). (B) Workflow for phosphoproteomic experiments of pig sinus node tissues. Proteins were extracted from anatomical sections of sinus node tissue, tryptic digests were multiplexed with TMTpro reagents, and phosphopeptides were enriched using TiO2 beads. The enriched peptides were fractionated at high pH and analysed by LC-MS/MS. (C) Heatmap of Pearson correlation coefficients for all measured phosphorylated peptide intensities across all samples. (D) Principal component analysis (PCA) of phosphorylated peptide intensities displaying separation of the samples according to treatment along the third component. (E) Gene set enrichment analysis (GSEA) using phosphorylation fold changes as input identified enriched pathways consequent to GLP-1 infusion. For multi-phosphorylated proteins, the greatest phosphorylation fold change was used. Enriched pathways are indicated in the plot; for all pathways, the adjusted P-value was below 1e−5. The gene set size refers to the number of genes covered in our data set of the respective pathways, and the colour indicates the normalized enrichment score (NES). All proteins measured in our dataset for the pathways highlighted in bold are visualized in the network in panel F. (F) Gene–KEGG pathway network (cnetplot) displaying the proteins contributing to the overrepresentation of the four pathways shown. Nodes are coloured by the greatest phosphorylation event fold change measured for the protein. Proteins known to be involved in heart rate regulation of the sinus node are encircled by a dashed line. (G) Western blot analysis of sinus node samples from pigs infused with GLP1 (n = 4) or vehicle (n = 4). Representative data of phospholamban phosphorylated at amino acid serine 16 [p-PLN(S16)] and loading control (GAPDH). Upper panel: Comparison of ratios of p-PLN(S16)/total PLN intensities. Lower panel: one-way Mann–Whitney U test. AU, arbitrary units.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/cardiovascres/120/12/10.1093_cvr_cvae120/1/cvae120f7.jpeg?Expires=1749995517&Signature=e-Yv9E4KRz3ifoI7cFlUWGXddsRqPT1f1liHk~50fwgLvoZ46g8iSZipA7urHg08XHuO-CkX6OTXFKu220gKL0nLB5M2shqMTnYvLzETwAeqK0z4ZyWncsC9P2GhEp4iFffNvYgcciGAJnHxirPWfAkIxyYauCIwAAeLgopWpyI8F3ZeqymLphNVa3WFtbohUbWCJKtDwqfISK9~Mo9Lygs~avkKwOisaNnhaYmpt3yKY0UWvsjvgs7ACrkhPDnmNufdUE2NH6A5da8lh6CzBUT4x6TNeNF6fZC5DmkiEDuWel0fa2CL~YzVkS1VWDLlAwuI4iXyY-FNaFghCcwG6A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Phosphoproteomics analysis suggests modulation of cAMP/Ca2+ signalling pathways in pig sinus node upon GLP-1 infusion. (A) Protocol of infusion of GLP-1 or vehicle prior to tissue collection (n = 4). (B) Workflow for phosphoproteomic experiments of pig sinus node tissues. Proteins were extracted from anatomical sections of sinus node tissue, tryptic digests were multiplexed with TMTpro reagents, and phosphopeptides were enriched using TiO2 beads. The enriched peptides were fractionated at high pH and analysed by LC-MS/MS. (C) Heatmap of Pearson correlation coefficients for all measured phosphorylated peptide intensities across all samples. (D) Principal component analysis (PCA) of phosphorylated peptide intensities displaying separation of the samples according to treatment along the third component. (E) Gene set enrichment analysis (GSEA) using phosphorylation fold changes as input identified enriched pathways consequent to GLP-1 infusion. For multi-phosphorylated proteins, the greatest phosphorylation fold change was used. Enriched pathways are indicated in the plot; for all pathways, the adjusted P-value was below 1e−5. The gene set size refers to the number of genes covered in our data set of the respective pathways, and the colour indicates the normalized enrichment score (NES). All proteins measured in our dataset for the pathways highlighted in bold are visualized in the network in panel F. (F) Gene–KEGG pathway network (cnetplot) displaying the proteins contributing to the overrepresentation of the four pathways shown. Nodes are coloured by the greatest phosphorylation event fold change measured for the protein. Proteins known to be involved in heart rate regulation of the sinus node are encircled by a dashed line. (G) Western blot analysis of sinus node samples from pigs infused with GLP1 (n = 4) or vehicle (n = 4). Representative data of phospholamban phosphorylated at amino acid serine 16 [p-PLN(S16)] and loading control (GAPDH). Upper panel: Comparison of ratios of p-PLN(S16)/total PLN intensities. Lower panel: one-way Mann–Whitney U test. AU, arbitrary units.
