Two‐Step Labeling of Endogenous Enzymatic Activities by Diels–Alder Ligation |
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Authors: | Lianne I Willems Martijn Verdoes Dr Bogdan I Florea Dr Gijsbert A van der Marel Prof Herman S Overkleeft Prof |
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Affiliation: | Leiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus Laboratories, Einsteinweg 55, 2333?CC Leiden (The Netherlands), Fax: (+31)?71‐5274307 |
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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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Keywords: | activity‐based protein profiling bioorthogonal chemistry cycloaddition hydrolases proteomics |
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