Extended Data Fig. 1: Development of the iPEX spatial proteomic analysis method.

a-b, Expected and observed m/z values of a standard peptide mix (a) or digested bovine serum albumin (b). c, Left, key steps of iPEX sample preparation process. Right, bright-field images of mouse retina at the states of pre-expansion, post-expansion with Coomassie blue staining, and post-expansion with hydrogel dehydrated on MALDI slide. ∅, abbreviated symbol for linear expansion factor (LEF); V, abbreviated symbol for area expansion factor (AEF). d, Workflow for processing MALDI-MSI data acquired via iPEX. e, The MS¹ spectra of expanded mouse retina iPEX sample. Right top, average ion intensity of iPEX detected peptides in mouse retina. f, Intensity distributions of top 30 most abundant peptide ions in mouse retina sections analysed by iPEX. g, Number of shared peptides and proteins in iPEX analysis of mouse retina sections. h, Venn diagram and scatter plot showing the overlap and correlation of proteins identified in three replicate mouse retina samples analyzed by iPEX at different EPS. i, Number of detected ions, peptides and proteins of MALDI analysis in mouse retina sections analysed via iPEX. j, Folds of relative enrichment and cross-pixel detection frequency of representative peptides from each layer of mouse retina. Scale bars indicated in images. INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, RGL: retinal ganglion layer, IPL: inner plexiform layer. The schematics in a, c and d were created using BioRender (https://www.biorender.com).