Fig. 2: Proteomic Landscape of EEA1-positive Endosomes in hESCs and iNeurons.

(A) Alterations in endosomal proteins during in vitro differentiation of hESCs to iNeurons, displayed as clusters identified based on patterns of expression, based on data from our previously reported TMT-based proteomic analysis of H9 cells using the NGN2 driver (31). (B) Analogous to panel A but for 4 markers of neurogenesis (SYP, GRIA2, NCAM1, and MAP2). (C) Schematic of our approach for quantitative analysis of the hESC and day 21 iNeuron endosomal proteome using the Endo-IP method for H9-E cells, and parental H9 cells as a control in triplicate or quadruplicate. Purified EEA1-positive membranes were eluted from the beads with a mild detergent, and proteins were digested with LysC and trypsin. The resulting peptides were labeled with 14-plex TMT and analyzed by mass spectrometry. (D) Violin plots of log₂ FC relative to WT (untagged control) cells for various groups of proteins found in Endo-IPs from hESCs or day 21 iNeurons. (E and F) Volcano plots of log₂ FC for tagged versus untagged (control) hESCs (panel E) or iNeurons (panel F) versus −log10(q-values) are plotted. Proteins annotated as lysosomal (cyan), plasma membrane (orange) or endosomal (green) are indicated. The dashed lines indicate the log₂ FC cut-off of 1.0 and −log10(q-value) cut-off of 2 (q-value of 0.01). (G) Comparison of proteins identified by Endo-IP in hESCs and day 21 iNeurons (upper Venn diagram). Pie charts display the proportion of identified proteins with abundance changes in either the hESC or iNeuron state, based on (31). The number of proteins present in SynGO are indicated.