Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS

Nan Li; Chi-Lin Kuo; Guillem Paniagua; Hans Van Den Elst; Martijn Verdoes; Lianne I. Willems; Wouter A. Van Der Linden; Mark Ruben; Eric Van Genderen; Jacob Gubbens; Gilles P. Van Wezel; Herman S. Overkleeft; Bogdan I. Florea

doi:10.1038/nprot.2013.065

Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS

Nan Li, Chi-Lin Kuo, Guillem Paniagua, Hans Van Den Elst, Martijn Verdoes, Lianne I. Willems, Wouter A. Van Der Linden, Mark Ruben, Eric Van Genderen, Jacob Gubbens, Gilles P. Van Wezel, Herman S. Overkleeft, Bogdan I. Florea^*

^*Corresponding author for this work

Chemistry

Research output: Contribution to journal › Article › peer-review

Abstract

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2-3 d, and approximately 8-10 d are needed to complete the entire protocol.

Original language	English
Pages (from-to)	1155-1168
Number of pages	14
Journal	Nature Protocols
Volume	8
Issue number	6
DOIs	https://doi.org/10.1038/nprot.2013.065
Publication status	Published - Jun 2013

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Li, N., Kuo, C.-L., Paniagua, G., Van Den Elst, H., Verdoes, M., Willems, L. I., Van Der Linden, W. A., Ruben, M., Van Genderen, E., Gubbens, J., Van Wezel, G. P., Overkleeft, H. S., & Florea, B. I. (2013). Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS. Nature Protocols, 8(6), 1155-1168. https://doi.org/10.1038/nprot.2013.065

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abstract = "Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2-3 d, and approximately 8-10 d are needed to complete the entire protocol.",

author = "Nan Li and Chi-Lin Kuo and Guillem Paniagua and {Van Den Elst}, Hans and Martijn Verdoes and Willems, {Lianne I.} and {Van Der Linden}, {Wouter A.} and Mark Ruben and {Van Genderen}, Eric and Jacob Gubbens and {Van Wezel}, {Gilles P.} and Overkleeft, {Herman S.} and Florea, {Bogdan I.}",

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AU - Verdoes, Martijn

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AU - Van Der Linden, Wouter A.

AU - Ruben, Mark

AU - Van Genderen, Eric

AU - Gubbens, Jacob

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AU - Overkleeft, Herman S.

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Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS

Abstract

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