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Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome

Abstract

Lactylation was initially discovered on human histones. Given its nascence, its occurrence on nonhistone proteins and downstream functional consequences remain elusive. Here we report a cyclic immonium ion of lactyllysine formed during tandem mass spectrometry that enables confident protein lactylation assignment. We validated the sensitivity and specificity of this ion for lactylation through affinity-enriched lactylproteome analysis and large-scale informatic assessment of nonlactylated spectral libraries. With this diagnostic ion-based strategy, we confidently determined new lactylation, unveiling a wide landscape beyond histones from not only the enriched lactylproteome but also existing unenriched human proteome resources. Specifically, by mining the public human Meltome Atlas, we found that lactylation is common on glycolytic enzymes and conserved on ALDOA. We also discovered prevalent lactylation on DHRS7 in the draft of the human tissue proteome. We partially demonstrated the functional importance of lactylation: site-specific engineering of lactylation into ALDOA caused enzyme inhibition, suggesting a lactylation-dependent feedback loop in glycolysis.

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Fig. 1: Discovery of a cyclic immonium ion of lactyllysine formed during MS/MS.
Fig. 2: Benchmarking the CycIm ion of lactyllysine as a marker of protein lactylation using model proteins and affinity-enriched lactylproteome.
Fig. 3: Rich nuclear and cytoplasmic nonhistone protein lactylation in the affinity-enriched lactylproteome of cultured human cells.
Fig. 4: Mining the Meltome Atlas showed that glycolytic enzymes in human cells are heavily lactylated.
Fig. 5: Unnatural amino acid-based mutagenesis and thermal shift assays demonstrated that certain lactylations are functionally important.
Fig. 6: Mining the draft map of the human proteome identifies widespread lactylation on DHRS7.

Data availability

The reprocessed SILAC-based lactylproteome datasets from human MCF-7 cells, the affinity-enriched lactylproteome from the plant fungal pathogen Botrytis cinerea, the affinity-enriched lactylproteome from the protozoan parasite Trypanosoma brucei, the draft map of the Human Proteome and the Meltome Atlas were accessed through the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) with the dataset identifier PXD014870, PXD020746, PXD023011, PXD000561 and PXD011929, respectively. Experimental data collected in this study can be accessed through the Consortium via the iProX partner repository40 with the dataset identifier PXD028488. Uncropped scans of immunoblots and gels shown in Fig. 5d and Supplementary Figs. 7d and 8a can be accessed via https://data.mendeley.com/datasets/4k9y97s7z9/1 on Mendeley Data. Source data are provided with this paper.

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Acknowledgements

This research was supported by the National Natural Science Foundation of China (grant 82173783 to H.Y., grants 81930109, 81720108032 to H.H. and grant 82104050 to N.W.), the National Key Research and Development Program of China (2021YFA1301300 to H.H.), the Fundamental Research Funds for the Central Universities (2632022YC03 to H.Y.), the Natural Science Foundation of Jiangsu Province (BK20210692 to N.W.), the Overseas Expertise Introduction Project for Discipline Innovation (G20582017001 to H.H.) and Sanming Project of Medicine in Shenzhen (SZSM201801060 to H.H.). We thank B. Shan, W. Chen and W. Li from PEAKS Studio and S. Sun from Northwest University for useful discussions.

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Contributions

H.Y., H.H., G.W. and Nanxi Wang conceived the project. N. Wan, Nian Wang, H.Y., H.H. and Nanxi Wang designed the experiments and analyzed the data. N. Wan, Nian Wang, S.Y., H.Z., Y.K. performed the proteomics experiments. D.W. established lactylation methods. S.Y. and L.L. contributed to data analysis. Nanxi Wang, R.T., S.T., H.L. and W.L. contributed to genetic code expansion and plasmid construction. X.W. and C.S. contributed to metabolomics experiments. H.Y., H.H., G.D., N. Wan and Nanxi Wang wrote the manuscript.

Corresponding authors

Correspondence to Nanxi Wang, Haiping Hao or Hui Ye.

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Nature Methods thanks W. Andy Tao and the other, anonymous, reviewers for their contribution to the peer review of this work. Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary Figs. 1–9, Supplementary Tables 1–7, Supplementary Notes 1–5 and Supplementary References.

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Supplementary Tables 1–7

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Unprocessed Western Blots and/or gels

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Wan, N., Wang, N., Yu, S. et al. Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome. Nat Methods 19, 854–864 (2022). https://doi.org/10.1038/s41592-022-01523-1

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