Protein Profiling of Active Cysteine Cathepsins in Living Cells Using an Activity-Based Probe Containing a Cell-Penetrating Peptide

Cell-permeable activity-based probes (ABPs) are capable of labeling
target proteins in living cells, thereby providing a powerful tool
for profiling active enzymes in their native environment. In this
study, we describe the synthesis and use of a novel trifunctional
cell-permeable activity-based probe (TCpABP) for proteomic profiling
of active cysteine cathepsins in living cells. We demonstrate that
although TCpABP contains cell-impermeable tags, it was able to enter
living cells efficiently via the delivery of a cell-penetrating peptide.
TCpABP also allowed simultaneous detection and affinity isolation
of labeled proteins with a fluorophore and a biotin motif, respectively.
We optimized the enrichment protocol to minimize contaminants and
identified 7 cathepsins, 2 of which have never been identified using
existing ABPs. We also used a label-free quantification approach to
quantify the relative abundances of active cathepsins and compared
them with their previously published mRNA expression levels. A high
degree of correlation between the mRNA expression levels and protein
relative activities was observed for most of the identified cathepsins
except cathepsin H. The results herein indicate that TCpABP is valuable
for the detection of active cathepsins in living cells and provides
useful guidelines for designing novel cell-permeable ABPs for in vivo labeling and their applications in in vivo proteomics studies.