Substrate specificity of mitochondrial intermediate peptidase analysed by a support-bound peptide library

•A synthetic support-bound FRET peptide library was constructed.•This was used to investigate the substrate specificity of recombinant human mitochondrial intermediate peptidase (hMIP).•Polar uncharged residues at P1 and P1′ are preferred by this enzyme.•hMIP can hydrolyse peptides shorter than 8 residues.•The importance of F/L/I at P8 and T/S/G at P5, in natural substrates of hMIP was not seen with this peptide library.