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.

Peptide peak lists were generated using MSConvert from the ProteoWizard Toolkit 32 , and data were searched against the 228-member peptide library using Protein Prospector software (v.5.17.0, University of California, San Francisco) 33 . Protein Prospector score thresholds were selected with a minimum protein score of 15 and minimum peptide score of 10. Maximum expectation values of 0.01 and 0.05 were used for protein and peptide matches, respectively. Substrate specificity profiles were generated with iceLogo software 18 using all possible cleavages in the MSP-MS library (n = 2,964) as the negative data set as described 16 . MS1 extracted ion chromatograms for label-free quantitation of select substrate and product species were generated using Skyline software (v.3.5, University of Washington) 34 . Specificity constants (k cat /K m ) were calculated as described 16 . All raw spectrum (.RAW) files from MSP-MS experiments in this study are available at the ProteoSafe resource (ftp:// /; username MSV000080171, password: hAPN).