Fig. 1

Phosphoproteome profiling of BMP9 and BMP10 stimulation in human umbilical vein endothelial cells (HUVECs). A Graphical representation of the experimental workflow for phosphoproteomic analysis. (1) HUVECs were stimulated or not (NS) with 10 ng/mL of BMP9 or BMP10 for 30 min. (2) Lysates from five biological replicates per condition were prepared and (3) subjected to reduction and alkylation, followed by (4) digestion using a combination of two endoproteinases, LysC and trypsin. (5) The resulting peptides were labeled with tandem mass tag (TMT) reagents and pooled for subsequent analysis. Phosphorylated peptides were then enriched using titanium dioxide (TiO2) beads (6.1), while a small portion of the pooled samples was reserved for proteomic analysis (6.2). (7, 8) The proteome and phosphoproteome samples were fractionated and analyzed using liquid chromatography tandem Mass spectrometry (LC–MS/MS). Phosphoproteomic analysis was performed twice on two separate fractions generating two technical replicates. (9–11) Data analysis was then performed using different bioinformatics tools. B Western blotting analysis of two biological replicates for each condition (NS, BMP9 or BMP10) used for phosphoproteomic analyses showing the levels of SMAD1/5 phosphorylation and ID1 expression. C Upper numbers represent count of identified and quantified phosphosites and their corresponding phosphoproteins annotated in UniProtKB database, as well as that of proteins (proteomic analysis) across all samples. Bottom numbers represent total count of differentially phosphorylated sites (DPS) and proteins by both BMP9 and BMP10