Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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 ( 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 on Mendeley Data. Source data are provided with this paper.


  1. Zhang, D. et al. Metabolic regulation of gene expression by histone lactylation. Nature 574, 575–580 (2019).

    CAS  Article  Google Scholar 

  2. Li, L. et al. Glis1 facilitates induction of pluripotency via an epigenome-metabolome-epigenome signalling cascade. Nat. Metab. 2, 882–892 (2020).

    CAS  Article  Google Scholar 

  3. Weinert, B. T. et al. Time-resolved analysis reveals rapid dynamics and broad scope of the CBP/p300 acetylome. Cell 174, 231–244 (2018).

    CAS  Article  Google Scholar 

  4. Gao, M., Zhang, N. & Liang, W. Systematic analysis of lysine lactylation in the plant fungal pathogen Botrytis cinerea. Front. Microbiol. 11, 594743 (2020).

    Article  Google Scholar 

  5. Zhang, N. et al. Protein lactylation critically regulates energy metabolism in the protozoan parasite Trypanosoma brucei. Front. Cell Dev. Biol. 9, 719720 (2021).

    Article  Google Scholar 

  6. Meng, X., Baine, J. M., Yan, T. & Wang, S. Comprehensive analysis of lysine lactylation in rice (Oryza sativa) grains. J. Agric. Food Chem. 69, 8287–8297 (2021).

    CAS  Article  Google Scholar 

  7. Chalkley, R. J. When target-decoy false discovery rate estimations are inaccurate and how to spot instances. J. Proteome Res. 12, 1062–1064 (2013).

    CAS  Article  Google Scholar 

  8. Anapindi, K. D. B., Romanova, E. V., Southey, B. R. & Sweedler, J. V. Peptide identifications and false discovery rates using different mass spectrometry platforms. Talanta 182, 456–463 (2018).

    CAS  Article  Google Scholar 

  9. Kim, M. S., Zhong, J. & Pandey, A. Common errors in mass spectrometry-based analysis of post-translational modifications. Proteomics 16, 700–714 (2016).

    CAS  Article  Google Scholar 

  10. Morelle, W. & Michalski, J. C. Analysis of protein glycosylation by mass spectrometry. Nat. Protoc. 2, 1585–1602 (2007).

    CAS  Article  Google Scholar 

  11. Bodenmiller, B., Mueller, L. N., Mueller, M., Domon, B. & Aebersold, R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat. Methods 4, 231–237 (2007).

    CAS  Article  Google Scholar 

  12. Olsen, J. V. et al. Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 4, 709–712 (2007).

    CAS  Article  Google Scholar 

  13. Trelle, M. B. & Jensen, O. N. Utility of immonium ions for assignment of ε-N-acetyllysine-containing peptides by tandem mass spectrometry. Anal. Chem. 80, 3422–3430 (2008).

    CAS  Article  Google Scholar 

  14. Potel, C. M., Lin, M. H., Heck, A. J. R. & Lemeer, S. Widespread bacterial protein histidine phosphorylation revealed by mass spectrometry-based proteomics. Nat. Methods 15, 187–190 (2018).

    CAS  Article  Google Scholar 

  15. Lassak, J. et al. Arginine-rhamnosylation as new strategy to activate translation elongation factor P. Nat. Chem. Biol. 11, 266–270 (2015).

    CAS  Article  Google Scholar 

  16. Zolg, D. P. et al. ProteomeTools: systematic characterization of 21 post-translational protein modifications by liquid chromatography tandem mass spectrometry (LC–MS/MS) using synthetic peptides. Mol. Cell Proteom. 17, 1850–1863 (2018).

    CAS  Article  Google Scholar 

  17. Muroski, J. M., Fu, J. Y., Nguyen, H. H., Ogorzalek Loo, R. R. & Loo, J. A. Leveraging immonium ions for targeting acyl-lysine modifications in proteomic datasets. Proteomics 21, e2000111 (2021).

    Article  Google Scholar 

  18. Wang, L., Brock, A., Herberich, B. & Schultz, P. G. Expanding the genetic code of Escherichia coli. Science 292, 498–500 (2001).

    CAS  Article  Google Scholar 

  19. Gaffney, D. O. et al. Non-enzymatic lysine lactoylation of glycolytic enzymes. Cell Chem. Biol. 27, 206–213 (2020).

    CAS  Article  Google Scholar 

  20. Zolg, D. P. et al. Building proteometools based on a complete synthetic human proteome. Nat. Methods 14, 259–262 (2017).

    CAS  Article  Google Scholar 

  21. Mongelard, F. & Bouvet, P. Nucleolin: a multiFACeTed protein. Trends Cell Biol. 17, 80–86 (2007).

    CAS  Article  Google Scholar 

  22. Jarzab, A. et al. Meltome atlas-thermal proteome stability across the tree of life. Nat. Methods 17, 495–503 (2020).

    CAS  Article  Google Scholar 

  23. Huang, J. X. et al. High throughput discovery of functional protein modifications by hotspot thermal profiling. Nat. Methods 16, 894–901 (2019).

    CAS  Article  Google Scholar 

  24. Zhang, Z. J., Pedicord, V. A., Peng, T. & Hang, H. C. Site-specific acylation of a bacterial virulence regulator attenuates infection. Nat. Chem. Biol. 16, 95–103 (2020).

    Article  Google Scholar 

  25. Kim, M. S. et al. A draft map of the human proteome. Nature 509, 575–581 (2014).

    CAS  Article  Google Scholar 

  26. Zemanová, L., Kirubakaran, P., Pato, I. H., Štambergová, H. & Vondrášek, J. The identification of new substrates of human DHRS7 by molecular modeling and in vitro testing. Int. J. Biol. Macromol. 105, 171–182 (2017).

    Article  Google Scholar 

  27. Moreno-Yruela, C. et al. Class I histone deacetylases (HDAC1-3) are histone lysine delactylases. Sci. Adv. 8, eabi6696 (2022).

    CAS  Article  Google Scholar 

  28. Wang, M. & Lin, H. Understanding the function of mammalian sirtuins and protein lysine acylation. Annu. Rev. Biochem. 90, 245–285 (2021).

    CAS  Article  Google Scholar 

  29. Huang, H. et al. Landscape of the regulatory elements for lysine 2-hydroxyisobutyrylation pathway. Cell Res. 28, 111–125 (2018).

    CAS  Article  Google Scholar 

  30. Bian, Y. et al. Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS. Nat. Commun. 11, 157 (2020).

    CAS  Article  Google Scholar 

  31. Meier, F. et al. Online parallel accumulation-serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer. Mol. Cell. Proteom. 17, 2534–2545 (2018).

    CAS  Article  Google Scholar 

  32. Frese, C. K. et al. Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos. J. Proteome Res. 10, 2377–2388 (2011).

    CAS  Article  Google Scholar 

  33. Bekker-Jensen, D. B. et al. An optimized shotgun strategy for the rapid generation of comprehensive human proteomes. Cell Syst. 4, 587–599 (2017).

    CAS  Article  Google Scholar 

  34. Costa Leite, T., Da Silva, D., Guimarães Coelho, R., Zancan, P. & Sola-Penna, M. Lactate favours the dissociation of skeletal muscle 6-phosphofructo-1-kinase tetramers down-regulating the enzyme and muscle glycolysis. Biochem. J. 408, 123–130 (2007).

    CAS  Article  Google Scholar 

  35. Prus, G., Hoegl, A., Weinert, B. T. & Choudhary, C. Analysis and interpretation of protein post-translational modification site stoichiometry. Trends Biochem. Sci. 44, 943–960 (2019).

    CAS  Article  Google Scholar 

  36. Beausoleil, S. A., Villén, J., Gerber, S. A., Rush, J. & Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292 (2006).

    CAS  Article  Google Scholar 

  37. Ma, B. et al. PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 17, 2337–2342 (2003).

    CAS  Article  Google Scholar 

  38. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

    CAS  Article  Google Scholar 

  39. Franken, H. et al. Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry. Nat. Protoc. 10, 1567–1593 (2015).

    CAS  Article  Google Scholar 

  40. Ma, J. et al. iProX: an integrated proteome resource. Nucleic Acids Res. 47, D1211–D1217 (2019).

    Article  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

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.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

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

Reporting Summary

Supplementary Table

Supplementary Tables 1–7

Source data

Source Data Fig. 5

Unprocessed Western Blots and/or gels

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing