Supplementary Figure 5: HR-MS and RP-HPLC analysis of biotin-labeled legumain activity containing unnatural amino acids.
From: Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity
Probe was synthesized according to the strategy presented in the main protocol (Block C, steps 138-151), and purified using reverse phase high performance (pressure) liquid chromatography (RP-HPLC) Waters system with semi-preparative C8 column. The purity of the probe was confirmed using analytical HPLC with C8 column (UV detector, 220nm). The molecular mass of the substrate was confirmed using High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI) and Time of Flight (TOF) module. Since the probe is more hydrophobic than the substrate, we used C8 (instead of C18) column to purify and analyze.
