The Rational Design of Therapeutic Peptides for Aminopeptidase N using a Substrate-Based Approach

Joshi, Shilvi; Chen, Lang; Winter, Michael B.; Lin, Yi-Lun; Yang, Yang; Shapovalova, Mariya; Smith, Paige M.; Liu, Chang; Li, Fang; LeBeau, Aaron M.

To determine the substrate specificity of APN, recombinant human APN (hAPN) was profiled using an unbiased and global substrate profiling approach referred to as Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) 16 . The MSP-MS assay uses a 228-member library of 14-mer synthetic and unmodified peptide substrates that were rationally designed to maximize physicochemical diversity within a small sequence space 17 . For specificity determination, hAPN was incubated with the MSP-MS peptide library and time-dependent peptide cleavage products were identified with liquid chromatography tandem mass spectrometry (LC-MS/MS). Statistical analysis that considers both cleaved and uncleaved positions in the peptide library 18 was subsequently performed to construct an iceLogo representation of hAPN P1-P4′ specificity as well as a corresponding heat map based on comprehensive Z-scores for each position (Fig. 1A ,B, and Supplemental Figure 1 ).