Fig. 4: Quantitative phosphoproteomics and enrichment analyses show EphA2 modulates phosphorylation of cell-cell adhesion and cytoskeletal proteins.
a Schematic representation of phosphoproteomic workflow. PC-3 cells were differentially labeled with light (12C) and heavy (13C) Arg and Lys isotopes for 6 passages, then activated with clustered ephrin-A1-Fc (efnA1-Fc) or treated with clustered Fc control for 20 min. Following cell lysis and clean-up, equal amounts of lysates from each condition were combined and digested with LysC and trypsin. Phosphopeptides were enriched with TiO2 and analyzed by LC-MS/MS. N = 4 independently SILAC-labeled, stimulated and processed biological replicates. The light and heavy amino acid labeling was alternated between replicates (“label swap”): replicates 1 and 3 were “forward labeled” (Fc control: light, ephrin-A1-Fc: heavy) and replicates 2 and 4 were “reverse labeled”. b Heat map showing the log2-transformed activated:control ratios across the four replicates for the 30 most regulated phosphopeptides. c Regulated phosphoproteins were categorized into protein classes according to function by manually curating information from HRPD, Panther and UniProt databases and the literature. GO enrichment analysis of Cellular Compartment (d) and Biological Process and Molecular Function terms (e). The ClueGo app for Cytoscape was used to calculate enrichment of terms associated with EphA2-regulated proteins compared to our own background dataset and to cluster related and redundant terms. Enrichment was evaluated using one-sided hypergeometric testing. FDR was controlled using the Benjamini–Hochberg correction for multiple hypothesis testing. Node size reflects the number of regulated phosphoproteins linked to a particular term; node color indicates the Benjamini–Hochberg corrected p value as a measure of significance as indicated.
