Protease-responsive mass barcoded nanotranslators for simultaneously quantifying the intracellular activity of...

Quantitatively delineating the activation network of multiple proteases that participate in cellular processes is highly essential for understanding the physiological and pathological states of cells. In this study, protease-responsive mass barcoded nanotranslators (PRMNTs) were engineered for revealing the activity of cascaded caspases in apoptosis in a multiplex and quantitative manner. In the PRMNTs, a series of mass tag-decorated gold nanoparticles (AuNPs) were tethered onto magnetic Fe₃O₄ nanospheres via a linker containing the substrate peptide of the target protease to form a "one-to-many" core-satellite structure. This nanostructure was internalized into the cells, underwent an enzymatic reaction within the cells, and allowed post-reaction mass spectrometry (MS) interrogation after magnetic separation from the cells. In the presence of intracellular caspases, enzymatic cleavage of the linker could be translated to the decreased ion signals of the mass tags on the remaining AuNPs in the PRMNTs by MS decoding. Benefiting from the multiplexing capability of MS, the intracellular activity of caspase-3, -8 and -9 that orchestrate the apoptotic process was simultaneously quantified at any given time. Kinetic analysis of caspase activity under stimulation of diverse anticancer drugs revealed that programmed cell death followed individual apoptosis pathways, differing in the activation degree and sequence of the caspase cascade. This work represents a modality that interfaces nanotechnology with MS for quantitatively probing the intracellular activity of multiple proteases, which opens up new avenues for revealing the apoptosis mechanism and developing innovative drugs.