Isolated human mitochondria underwent DSSO crosslinking. Following treatment, the mitochondria were separated into soluble and insoluble fractions. Both fractions were then subjected to multidimensional fractionation, including SCX and hSAX. The fractionated peptides were subsequently analyzed using CL-MS. FDR-based error estimation facilitated the creation of a comprehensive mitochondrial interaction network, revealing novel protein-protein interactions.