Extended Data Fig. 2: iPEX enables identification of colocalized protein groups and extra-somatic structures in the mouse retina and tandem MS validations of selected peptide ions.

a-b, Ion information (a) and the MS² spectra of the matched fragment ions acquired on the timsTOF fleX MALDI-2 platform (b) for 10 selected proteolysis-dependent precursor ions of which the MS² spectra are matching with the MS¹ annotated peptide sequences. c, Immunofluorescence (IF) staining of Dync1h1 and Sag expression in mouse retina tissues. d, Intensity distributions of indicated peptide ions in conventional MALDI and iPEX analysed mouse retina segments. Lines inside each violin represents quantile intensity value cross pixels. e-f, Correlation coefficient analysis of all annotated peptide ions (e) and top colocalized peptides (f) with distributions of the target ion (m/z 1,607.959) in the mouse retina iPEX sample. g, Ion intensity maps of the target ion and some top colocalized peptides. h-i, Correlation coefficient analysis of all annotated peptide ions (h) and top colocalized peptides (i) with distributions of the target ion (m/z 1,578.791) in the mouse retina iPEX sample. j, Ion intensity maps of some top colocalized peptides. k, IF image of Cltc expression in mouse retina tissues. l, Ion intensity maps of indicated peptide ions enriched in the neural synapse layers (OPL and IPL). The black arrow points to the outer plexiform layer. m, Experimental procedure evaluating the dynamic range of iPEX protein detection using the protein extracts from fetal bovine serum (FBS). n, Number of detected peptides and proteins in precipitated and serial-diluted FBS proteins analysed via iPEX or conventional MALDI-MSI. o, Average ion intensity (solid line) and standard deviation (in the background) of peptides detected in precipitated FBS protein analyzed via iPEX. Scale bars indicated in images. The schematic in m was created using BioRender (https://www.biorender.com).