Fig. 2: Landscape of the early/sorting endosome proteome.

a Schematic depicting an approach for detailed analysis of the early endosome proteome based on Endo-IP. Cells were employed in biological quadruplicate using 293 cells as a control (n = 4). Immune complexes were digested with trypsin, peptides labeled using 8-plex TMT and analyzed by mass spectrometry. b Volcano plot for quadruple Endo-IP associated proteins relative to control cells lacking tagged EEA1. Two-sided Student’s t-test was performed and adjusted for multiple comparisons by two-stage Benjamini & Hochberg step-up procedure. The dashed lines indicate threshold of Log₂FC > 1.0 with p value less than 0.02. Selected proteins linked physically or functionally with early endosomes are shown in red. This experiment employed 293^EL cells (clone 33). c Box plots depicting the enrichment of various classes of proteins based on the annotation of Itzhak et al.³² demonstrating that proteins assigned to endosomes and to a lesser extent, lysosomes are the most highly enriched, while ER, mitochondrial, and a subset of PM proteins are depleted. Left border, interior line, and right border in the box plot represent the 1st quartile, median, and 3rd quartile, respectively. Two-sided Student’s t-test was performed and adjusted for multiple comparisons by two-stage Benjamini & Hochberg step-up procedure. d Proteins significantly enriched in the Endo-IP are organized into functional modules. All the proteins shown were identified as being enriched with Log₂FC > 1.0 in the FLAG-EEA1 sample, with the exception of WASHC2A (indicated in italic) which had a Log₂FC value of 0.99. Dotted lines for the early endosome RAB network indicate the presence of physical interactions or association based on proximity biotinylation experiments. Proteins indicated with an asterisk were found in common with the endosomal compartment proteome reported by Ihtzak et al.³². See Supplementary Data 3. e Comparison of proteins enriched in Endo-IP with the endosomal proteome identified by Ihtzak et al, 2016 using gradient fractionation³². Two-sided Student’s t-test was performed and adjusted for multiple comparisons by two-stage Benjamini & Hochberg step-up procedure. Proteins (262) in the very high, high, medium, and low confidence intervals from HeLa cells³² and 316 proteins from the Endo-IP from 293^EL cells (Log₂FC > 1.0) were used, with the exception of proteins with a red asterisk which were just below the fold change cut-off. Proteins overlapping in the two data sets are indicated with asterisks in panel d.