Murine podocyte mitochondrial complexsome analysis

Proximity labeling discovered a few mitochondrial cristae organizing proteins as NDUFS4 interacting protein. However proximity labeling is unable to differentiate between a direct interaction or a mere close association between NDUFS4 and cristae forming proteins. Additionally, it does not specify if the association of NDUFS4 with these proteins occurs in the context of individual complexes or RSCs. Consequently, to explore whether these cristae forming proteins comigrate and are retained within different complexes in RSCs, we performed complexsome profiling analysis on isolated mitochondria from HG-treated Ndufs4 overexpressed podocytes, with HG-treated cells as control. We separated ETC complexes with BN-PAGE, sliced five distinct bands representing distinct RSCs, labeled 1–5, after Coomassie blue staining, followed by mass spectrometry (LC-MS/MS).