Chemical probes for spatially resolved measurement of active enzymes in single cells

Ongoing advances in chemical proteomic methods have facilitated detection and quantification of enzymatic activity, a highly informative parameter that is not captured in protein abundance measurements. However, some biological questions remain unanswered, since current gel- or LC-MS/MS-based detection methods suffer from limitations stemming from sample homogenization, signal-averaging, and an inherent bias toward abundant proteins. To address these shortcomings, we recently developed an activity-based proximity ligation (ADPL) platform to capture and quantify enzyme activity on the level of single cells, with high intra- and intercellular spatial resolution. In this chapter, we briefly discuss the rationale behind the ADPL platform, the design transition from the initial "sandwich-complex" workflow to the optimized, "direct conjugate" ADPL method, and conclude with detailed protocols for each. We also describe our novel use of the homo-bifunctional linker, disuccinimidyl suberate (DSS), to conjugate proteins and oligonucleotides, thus generating the necessary antibody-oligonucleotide recognition reagents for ADPL. Finally, we demonstrate the utility of ADPL to characterize enzyme activity from cytosol to nucleus, and specifically detect enzyme activity using "direct conjugate" ADPL.