Abstract
A long-standing goal in biomedical research is to label and manipulate intracellular targets, which could be achieved through the cytosolic delivery of exogenous functional proteins. The development of Tat clusters has advanced the nontoxic intracellular delivery of functional antibodies at low concentrations, but the variety of proteins that can be successfully delivered remains limited. Here, we find that by simply reversibly modifying the surface of functional proteins with anionic peptide patches, various protein cargoes (which are normally difficult to deliver) can be delivered into living cells by synergetic electrostatic interactions with the cationic cell-penetrating peptide clusters TAT3. To demonstrate the applicability of this approach, we successfully deliver functional proteins with widely varying molecular weights (∼1.5 kDa to 430 kDa) and isoelectric points (less than 5 to greater than 9) into the cytosol of cells. By exploiting this method, we also achieve protein delivery in plant tissues, which is more challenging due to the presence of intact plant cell walls. This strategy is further applied for the cytosolic delivery of synthetic protein probes carrying posttranslational modifications (PTMs), which can aid in in situ mapping of the intracellular PTM-mediated interactome. Overall, this strategy is expected to enrich cytosolic protein delivery technology and help to repurpose a wide range of customized and therapeutic proteins for emerging intracellular applications.
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Data availability
The mass spectrometry proteomics data generated in this study have been deposited in the ProteomeXchange [http://proteomecentral.proteomexchange.org] database under accession code PXD059494. Source data are provided with this paper. Data supporting the findings of this study are available from the corresponding author Yi-Ming Li upon request.
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Acknowledgements
This project was supported by the National Key R&D Program of China (No. 2022YFC3401500 for L. Liu), and NSFC (Nos. 22277020, 22227810 for Y.-M. Li, 22137005, 92253302 for L. Liu, 22377117 for J. Shi), and Anhui Provincial Natural Science Foundation (No. 2508085JX004 for Y.-M. Li), and the Fundamental Research Funds for the Central Universities (PA2024GDGP0037, JZ2024YQTD0600 for Y.-M. Li), and Beijing Life Science Academy (BLSA, No: 2023000CC0130) and the XPLORER prize (for L. Liu).
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Conceptualization and Scientific direction: Y.-M. Li, and L. Liu. Writing: Y.-M. Li, L. Liu, X. Hua. Cell biological and fluorescence imaging experiments: X. Hua, Y. Guo. Protein modification and biochemical experiments: Y. Guo, X. Han, J. Shi. Peptide synthesis: P. Li, J. Chen, J. Li, G. Chu. MS sample preparation: X. Han, Y. Wang. MS data collection and analysis: Y. Wang, Y. Guo.
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Hua, X., Guo, Y., Li, P. et al. Reversible surface modifications of functional proteins for accelerated cytosolic delivery via cell-penetrating peptide clusters. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70054-6
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DOI: https://doi.org/10.1038/s41467-026-70054-6
