Polychrome activity profiling distinguishes Cys proteases and their isoforms in plants.

Polychrome activity profiling using E-64 warhead probe cocktails distinguishes papain-like cysteine proteases and their isoforms in plant crude proteomes, producing specific apparent colors.

• Why it matters: Activity-based profiling can characterize enzyme classes in complex mixtures, but differentiating closely related protease isoforms requires probes that separate labeling by affinity rather than just presence.
• What they did: The study used fluorescent E-64 warhead probe cocktails with different fluorophores linked by short linkers, then applied polychrome labeling to resolve protease families and RD21-like isoforms, supported by molecular modeling.
• The result: Polychrome labeling showed differential labeling of RD21-like isoforms with C-terminal granulin domains, and modeling suggested the granulin domain may form a fluorophore-binding pocket that influences substrate selectivity.

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