Two-step labeling of endogenous enzymatic activities by Diels-Alder ligation

Lianne I. Willems; Martijn Verdoes; Bogdan I. Florea; Gijsbert A. Van Der Marel; Herman S. Overkleeft

doi:10.1002/cbic.201000280

Two-step labeling of endogenous enzymatic activities by Diels-Alder ligation

Lianne I. Willems, Martijn Verdoes, Bogdan I. Florea, Gijsbert A. Van Der Marel, Herman S. Overkleeft

Chemistry

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

Abstract

A ligation strategy based on the Diels-Alder [4+2] cycloaddition for the two-step activity-based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene-derivatized proteasome probes was synthesized, along with a dienophile-functionalized BODIPY(TMR) tag. These probes were applied in a Diels-Alder labeling procedure that enabled us to label active proteasome β-subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene-derivatized cathepsin probe. Importantly, the Diels-Alder strategy described here is fully orthogonal with respect to the Staudinger-Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.

Original language	English
Pages (from-to)	1769-1781
Number of pages	13
Journal	Chembiochem
Volume	11
Issue number	12
DOIs	https://doi.org/10.1002/cbic.201000280
Publication status	Published - 16 Aug 2010

Keywords

Activity-based protein profiling
Bioorthogonal chemistry
Cycloaddition
Hydrolases
Proteomics

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10.1002/cbic.201000280

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title = "Two-step labeling of endogenous enzymatic activities by Diels-Alder ligation",

abstract = "A ligation strategy based on the Diels-Alder [4+2] cycloaddition for the two-step activity-based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene-derivatized proteasome probes was synthesized, along with a dienophile-functionalized BODIPY(TMR) tag. These probes were applied in a Diels-Alder labeling procedure that enabled us to label active proteasome β-subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene-derivatized cathepsin probe. Importantly, the Diels-Alder strategy described here is fully orthogonal with respect to the Staudinger-Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.",

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author = "Willems, {Lianne I.} and Martijn Verdoes and Florea, {Bogdan I.} and {Van Der Marel}, {Gijsbert A.} and Overkleeft, {Herman S.}",

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T1 - Two-step labeling of endogenous enzymatic activities by Diels-Alder ligation

AU - Willems, Lianne I.

AU - Verdoes, Martijn

AU - Florea, Bogdan I.

AU - Van Der Marel, Gijsbert A.

AU - Overkleeft, Herman S.

PY - 2010/8/16

Y1 - 2010/8/16

N2 - A ligation strategy based on the Diels-Alder [4+2] cycloaddition for the two-step activity-based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene-derivatized proteasome probes was synthesized, along with a dienophile-functionalized BODIPY(TMR) tag. These probes were applied in a Diels-Alder labeling procedure that enabled us to label active proteasome β-subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene-derivatized cathepsin probe. Importantly, the Diels-Alder strategy described here is fully orthogonal with respect to the Staudinger-Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.

AB - A ligation strategy based on the Diels-Alder [4+2] cycloaddition for the two-step activity-based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene-derivatized proteasome probes was synthesized, along with a dienophile-functionalized BODIPY(TMR) tag. These probes were applied in a Diels-Alder labeling procedure that enabled us to label active proteasome β-subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene-derivatized cathepsin probe. Importantly, the Diels-Alder strategy described here is fully orthogonal with respect to the Staudinger-Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.

KW - Activity-based protein profiling

KW - Bioorthogonal chemistry

KW - Cycloaddition

KW - Hydrolases

KW - Proteomics

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Two-step labeling of endogenous enzymatic activities by Diels-Alder ligation

Abstract

Keywords

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