Fig. 3: Proximity extracellular interactome of over-expressing EGFR by EGFR- FabID on cells.

a Workflow of PPI analysis targeting EGFR overexpression. LC-MS/MS identified 640 biotinylated peptides, of which 189 were selected as those with AGIA-FabID ratios >1 and P < 0.05. The number of peptides was then narrowed to 64 peptides that had transmembrane (TM) regions or contained “membranes” in the gene ontology (GO) term. 64 peptides were matched into 22 proteins. Twelve of the 22 proteins were known to interact with EGFR, and 10 were novel EGFR-interacting proteins. b Volcano blot (3 biological replicates) of peptides detected as biotinylated peptides by LC-MS/MS in (a). Peptides derived from cell membrane proteins are indicated by red dots. c Table of extracellular proteins identified by mass spectrometry (three biological replicates, EGFR-FabID/AGIA-FabID ratios >1 and P < 0.05). The number of DTX edges for each protein and EGFR are shown in the table. Proteins in black font are those already known to interact with EGFR, whereas those in blue represent unknown EGFR-interacting proteins. d Pathway analysis of extracellular proteins detected using mass spectrometry. The Gene Ontology software Drug Target Excavator (DTX) (https://harrier.nagahama-i-bio.ac.jp/dtx/) and IntAct database, including EGFR-interacting proteins (https:///ebi.ac.uk/intact/), were used to analyse protein interactions. b, c Significant changes in the volcano plots and heat map were calculated by Student’s two-sided t test and the false discovery rate (FDR)-adjusted P-values calculated using Benjamini–Hochberg method are shown in the Supplementary Data 1.