Extended Data Fig. 4: Assessments for the annotation accuracy, subcellular compartments, dynamic ranges and pixel distributions of iPEX detected proteins.

a, Left, Coomassie blue staining of post-expansion mouse cerebellum before and after microdissection on the LCM sample slide. Right, sample information for LCM-LC-MS/MS spatial proteomics. b, Distribution of ion-peptide matching count in indicated mouse cerebellum layers when using LCM- and bulk tissue-derived MS spectral libraries. c, Abundance of LCM–LC-MS/MS detected peptides in indicated regions of mouse cerebellum samples. Gray dots represent all proteins detected in LCM-LC-MS/MS and red dots represent protein detected in both LCM-LC-MS/MS and iPEX analysis. Box-and-whisker plot inside each plot indicates median value and range of corresponding scatter plot. d, Cumulative distribution function of overlapped peptides in LCM–LC-MS/MS and iPEX analysis across the abundance rank in LCM–LC-MS/MS. e, Top 10 abundant high-confidence (≥2 UPs) proteins in LCM library and iPEX analysis in mouse cerebellum. f, Percentage of iPEX or LC-MS/MS detected proteins belonging to the indicated gene ontology (GO) cellular compartment terms. g, Distribution of detected peptides and proteins (≥2 UPs) across pixels in the mouse retina iPEX sample, mouse brain iPEX sample, and human cerebral organoid iPEX sample, respectively. Box-and-whisker plot inside each violin plot indicates median and quantile value of corresponding scatter plot. h, Coomassie blue staining and bright-field images of mouse cerebral coronal sections. i, Average ion intensity of iPEX detected peptides in mouse brain samples. j, Estimated anatomical divisions suggested by the Allen Mouse Brain Atlas database³³ for the iPEX brain slice. Scale bars indicated in images. Brain images in j were reproduced from the Allen Mouse Brain Reference Atlas (https://mouse.brain-map.org)³³.