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.
Britton Chance Center for Biomedical Photonics, Department of Biomedical Engineering, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology (HUST), Wuhan, China.
Fengkai
F
Fan
Si
S
Nie
Eric B
EB
Dammer
Duc M
DM
Duong
Deng
D
Pan
Lingyan
L
Ping
Linhui
L
Zhai
Junzhu
J
Wu
Xuechuan
X
Hong
Lingsong
L
Qin
Ping
P
Xu
Yu-Hui
YH
Zhang
D000345:Affinity Labels / Q000737:chemistry; D000818:Animals; D002403:Cathepsins / Q000032:analysis* / Q000737:chemistry; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008264:Macrophages / Q000737:chemistry*; D051379:Mice; D018613:Microscopy, Confocal; D015335:Molecular Probes / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry; D060327:Solid-Phase Synthesis Techniques; D013379:Substrate Specificity
D000345:Affinity Labels; D057846:Cell-Penetrating Peptides; D005456:Fluorescent Dyes; D015335:Molecular Probes; D012333:RNA, Messenger; D002403:Cathepsins; D003545:Cysteine