Abstract
Integration of chemical probes into proteomic workflows enables the interrogation of protein activity, rather than abundance. Current methods limit the biological contexts that can be addressed due to sample homogenization, signal-averaging, and bias toward abundant proteins. Here we report a platform that integrates family-wide chemical probes with proximity-dependent oligonucleotide amplification and imaging to quantify enzyme activity in native contexts with high spatial resolution. Application of this method, activity-dependent proximity ligation (ADPL), to serine hydrolase and cysteine protease enzymes enables quantification of differential enzyme activity resulting from endogenous changes in localization and expression. In a competitive format, small-molecule target engagement with endogenous proteins in live cells can be quantified. Finally, retention of sample architecture enables interrogation of complex environments such as cellular co-culture and patient samples. ADPL should be amenable to diverse probe and protein families to detect active enzymes at scale and resolution out of reach with current methods.
Highlights
Integration of chemical probes into proteomic workflows enables the interrogation of protein activity, rather than abundance
activity-dependent proximity ligation (ADPL) integrates the activity-dependent and family-wide tagging of endogenous, active enzymes afforded by chemical probes, with the specific and robust signal amplification afforded by barcoded oligonucleotide proximity ligation and amplification (Fig. 1)[21]
Compared to existing activity-based proteomic approaches with gel or LC-MS/MS as a readout, the incorporation of a specific and robust amplification scheme applied in native cell environments allows for significant expansion of the questions that can be addressed in biological systems
Summary
Integration of chemical probes into proteomic workflows enables the interrogation of protein activity, rather than abundance. Activity-based proteomic technologies, on the other hand, integrate enzyme- or protein-family-specific chemical probes with traditional mass spectrometry or gel-based profiling methods in order to detect and quantify protein activity, rather than abundance[12,13,14]. These measurements can be made directly with complex samples such as lysate, tissues, and biological fluids to measure changes in protein activity, often for entire families of proteins of a 100 or more[15,16,17], that result from endogenous biological signals or the action of exogenous molecules (e.g., therapeutics). Small-molecule “turn-on” probes[29] typically lack the ability to provide precise spatial information due to signal diffusion, and sometimes do not reflect a Live cell treatment
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