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bioRxiv · 10.1101/2025.04.28.651067

Expanding and applying reactivity-based protein profiling (RBPP) in Arabidopsis

Mooney, B. C.·Lyu, J.·Kaschani, F.·Kaiser, M.·van der Hoorn, R. A. L.
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Abstract

Chemical probes that label surface-exposed amino acid residues in native proteomes serve as powerful tools for studying native reactivities, yet their use has primarily been limited to drug discovery. In this study, we expand the application of reactivity proteomics by monitoring ATP-induced changes over 21,000 reactive lysine, serine, threonine, and tyrosine residues across native Arabidopsis proteomes using biotinylated N-hydroxysuccinimide (BioNHS) ester probes. ATP caused significant differential labelling. Labelling was reduced in ATP-binding pockets, consistent with ligand occupancy, whereas many sites outside ATP-binding regions showed either reduced or increased labelling in response to ATP. Structural modelling revealed that these differentially labelled sites are associated with ATP-induced conformational changes, altering lysine exposure. Notable examples of ATP-driven structural shifts include adenylate kinase ADK4, acyl-activating enzyme AAE3, cell division control protein CDC48A and its interacting partner PUX1, and ATP-induced 26S proteasome assembly. Furthermore, we show that reactivity proteomics using phosphonic acid-NHS (PhoNHS) followed by IMAC enrichment offers a complementary and expanded list of labelling sites. Our findings establish reactivity proteomics as a versatile and powerful platform for exploring protein conformational dynamics, metabolite-protein interactions, and regulatory mechanisms directly in native proteomes.

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Mooney, B. C., Lyu, J., Kaschani, F., Kaiser, M., van der Hoorn, R. A. L.. 2025-04-30. Expanding and applying reactivity-based protein profiling (RBPP) in Arabidopsis. https://doi.org/10.1101/2025.04.28.651067

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