Synthesis and Application of an Activity‐Based Peptide‐Peptoid Hybrid Probe for the Immunoproteasome

The immunoproteasome (iCP), a specific isoform of the proteasome's catalytic particle, is becoming an important protein complex of interest in various diseases. However, there is still much left to be learned about its activity in cells and how this can be altered by various endogenous conditions or with treatment with small molecules. Current strategies to investigate the iCP lack in their ability to be used in live, intact cells, limiting them to use in endpoint experiments. The iCP-selective probe presented here has been shown to be compatible with various live-cell assays, including monitoring iCP activity kinetically in a plate reader-based assay and observing single cells with confocal microscopy. A well-studied iCP-selective inhibitor, ONX-0914, has also been demonstrated to decrease the fluorescence signal of the iCP probe in both of these assays, showing its potential function in investigating small-molecule modulators of the iCP. (c) 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of an immunoproteasome-selective peptide-peptoid hybrid probe Basic Protocol 2: Expression of the immunoproteasome in A549 cells Basic Protocol 3: Using the immunoproteasome probe to monitor activity in live cells with a fluorescence plate reader Basic Protocol 4: Using the immunoproteasome probe to monitor activity in live cells with confocal microscopy.