Supplementary Information

for

Grassystatin-derived peptides selectively inhibit cathepsin E and have low affinity to cathepsin D

Sophie Stotz a,b, Daniel Bleher a, Hubert Kalbacher c, Andreas Maurer abc

a Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University, Tübingen, Germanyb iFIT Cluster of Excellence, Eberhard Karls University, Tübingen, Germanyc Interfaculty Institute of Biochemistry, Eberhard Karls University Tübingen, Germany

Supplementary Fig. 1: ESI-MS spectra of the fluorogenic protease substrate (left graph) and of the biotinylated grassystatin-derived peptide (1, right graph).

Supplementary Fig. 2: ESI-MS spectra of the short acetylated grassystatin derivative (2, left graph) and of the biotinylated pepstatin-derived peptide (3, right graph).

Supplementary Fig. 3: SDS-PAGE of catD and catE isolated from overexpressing HEK293 cells used for the enzyme assays. The respective enzyme is marked with an asterix.

Supplementary Fig. 4: Peptide mass fingerprinting of the 65 kDa band from murine kidney (Fig. 3, lane 12) identified the protein as the alpha chain of propionyl-CoA carboxylase (PCCA).

Supplementary Fig. 5: Peptide mass fingerprinting of the 55 kDa band from murine kidney (Fig. 3, lane 12) identified the protein as the beta-chain of propionyl-CoA carboxylase (PCCB).

Supplementary Fig. 6: Peptide mass fingerprinting of the 35 kDa band from murine kidney (Fig. 3, lane 12) identified napsA (kidney-derived aspartic protease).

Supplementary Fig. 7: Peptide mass fingerprinting of the 37 kDa band from murine spleen (Fig. 3, lane 11) identified catE. Surprisingly, catD was also among the results, although with a lower score and sequence coverage.

Supplementary Fig. 8: Peptide mass fingerprinting of the 37 kDa band from murine stomach (Fig. 3, lane 7) identified catE.