Fig. 3: Validation and evaluation of the CAT-S system.

a–c CAT-S-enabled in situ mitochondrial proteomics of living HeLa, HEK293T, and K562cells (from top to bottom), treated with/without Ir1 and SF2 followed by blue LED irradiation. a Labeling signals measured by immunoblotting (n = 3 biological replicates). b Frequency distribution profiles of MS-identified proteins (≥2/3 replicates) with (red) and without (gray) mitochondrial annotation according to the combined human MitoCarta3.0 and UniProt database. A filter based on +/− catalyst ratio (set to 5.0 for the three cell lines) was further applied to subtract background noise (non-specific proteins). c ROC curves of CAT-S for detecting annotated mitochondrial proteins, with AUC (area under the curve) values shown. d Mitochondrial specificity analysis for HeLa, HEK293T, and K562 cells with the columns showing the fraction of proteome with prior mitochondrial annotation in the database. e Contribution of annotated mitochondrial proteins (red) in the total protein MS abundance. The estimated abundance of mitochondrial proteins in the whole cellular proteome is shown for comparison. f Comparison of mitochondrial proteomic coverage between this work and CAT-Prox. g Venn diagram showing the overlap between captured mitochondrial proteomes. h GO biological process enrichment analysis of the mutually captured mitochondrial proteome from HeLa, HEK293T, and K562. Top terms (P < 10⁻²⁰, cumulative hypergeometric test) were shown. i Sub-mitochondrial preference analysis of captured mitochondrial proteomes from HeLa, HEK293T, and K562 cells. Proteins were classified into “matrix*”, “intermembrane space (IMS)”, and “mitochondrial outer membrane (MOM)” according to MitoCarta3.0 database. The “matrix*” category also contains the matrix-facing subunits of inner membrane protein complexes according to the literature⁵. Source data are provided as a Source Data file. Created with BioRender.com (agreement number LJ26KFYU6T).