Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: Kinetics of intracellular Ca 2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB‐KO) were seeded on 96‐well plates, loaded with Fluo‐4 dye and stimulated with 100 nM EDN or PBS. Fluo‐4 fluorescence was monitored over 10 min ( n = 6). Representative example of three independent experiments. The error bars indicate ± standard deviation. Time course of AKT and CREB phosphorylation. Melanoma cell lines were stimulated with 100 nM EDN for 2–90 min. AKT and CREB phosphorylation and expression were evaluated by Western blot. Representative example of three independent experiments. Experiment design for the UACC257 phosphoproteomic study, showing time points, cell line derivatives, replicate structure and GIST composition. The GIST was generated by pooling all heavy SILAC plates (blue) and spiked into all UACC257 (red) and UACC257 EDNRB‐KO (green) samples. The A2058 cell line was also analysed in biological triplicates and a GIST but without EDNRB‐KO cells. High reproducibility in the UACC257 and A2058 data sets. CV distributions for triplicates were calculated and are provided in Fig D. All CV values were grouped according to cell line to represent data set‐specific CV distributions. Numbers and lines indicate the median.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Fluorescence, Standard Deviation, Phospho-proteomics, Expressing, Western Blot, Generated, Multiplex sample analysis

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: Validation of EDNRB ablation in UACC257 and A2058. EDNRB expression in WT and EDNRB‐KO cell lines was analysed by Western blot. Ten and 50 μg total protein were loaded for UACC257 and A2058, respectively. Specificity of TiO 2 enrichment, defined as percentage of phosphopeptide identifications over all identifications. The whiskers show 1.5*IQR (Tukey). LuciPHOr performance. Number of peptides with localised phosphates is compared to all phosphopeptides in the data set if phosphate positions were delocalised for different global FLR cut‐offs. CV of phosphopeptide quantification was benchmarked in six process replicates ( n = 6) of the full workflow. Peptides were quantified from the same result files using LFQ (Progenesis QI for proteomics) or the Skyline SILAC GIST workflow. CV violin plots for benchmarking samples and each triplicate in the UACC257 and A2058 data sets are shown. Lines and numbers indicate the medians for each group. Proportions of singly, doubly and triply phosphorylated peptides for each cell line. Proportions of phosphorylated amino acid residues for each data set.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Biomarker Discovery, Expressing, Western Blot, Phospho-proteomics, Targeted Proteomics, Multiplex sample analysis

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA. Colours indicate experimental groups, and shading is mapped to time. Overlap between UACC257 and A2058 data sets. From top to bottom: Identified proteins, identified phosphopeptides, EDN‐regulated phosphopeptides at q < 0.1, and q < 0.1 & FC > 1.5 up or down. Comparison of the kinetics of the 242 shared target phosphopeptides. Log 2 FC of EDN/Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Phospho-proteomics, Comparison

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: Hierarchical clustering of phosphopeptide MS1 intensity for UACC257 separates samples according to the presence and activation of the EDNRB receptor. Clustering was performed on the UACC257 data set in R following log 2 transformation of normalised MS1 intensities, imputation of missing values with the imputeLCMD R package and z ‐score transformation. PCA of the A2058 data set. Phosphopeptide MS1 intensities in A2058 samples were analysed by PCA using R. Colours indicate experimental groups, and shading is mapped to time. Examples kinetics of known (ERK1 T202/Y204) and novel phosphosites (MEK2 S23 & ROCK2 S1134/S1137) on relevant kinases in UACC257 (top) and A2058 (bottom) cells from phosphoproteomic data sets. Data are represented as mean ± SD. * q < 0.1. Two sided unpaired t ‐tests followed by Benjamini‐Hochberg multiple testing correction ( n = 3).

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Phospho-proteomics, Activation Assay, Transformation Assay

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: A, B EDN phosphoproteomic data were analysed in IPA. Significance of term enrichment is indicated through shading as −log ( P ‐value). Regulated pathways belonging to the classes “Canonical Pathways” (A) and “Disease and Biofunction” (B) with significant enrichment ( P < 10 −3 ) are shown for UACC257 and A2058. Regulated terms were grouped according to highest order ontology terms, which are shown on the left.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques:

Journal: Molecular Systems Biology

Article Title: Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data

doi: 10.15252/msb.20198828

Figure Lengend Snippet: A, B EDN target phosphopeptides for (A) UACC257 and (B) A2058 were segregated into six clusters each, using c‐means clustering (MFuzz). Each line represents one phosphopeptide, and cluster membership is indicated by colour. Most likely kinases for each phosphosite were predicted using NetworKIN, and enrichment of kinase substrates was calculated for each cluster. Enriched kinases are indicated above each cluster. Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster. Detailed information on the kinase enrichment is provided in Appendix Table S1 . C Average MS1 intensity z ‐score kinetics for 217 predicted CaMKII‐α sites that are shared between both cell lines. For comparison, average ( n = 6) calcium abundance kinetics from Fig A are shown.

Article Snippet: A2058 EDNRB knockout cells (ENDRB‐KO) were generated using the TALEN technology (Cellectis).

Techniques: Phospho-proteomics, Comparison