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

Lactylation is a post translation modification (PTM) derived from lactate and was previously shown on lysine residues of human histones. Importantly, Zhang, et al identified histone lactylation as an epigenetic change that drives M1 to M2 macrophage polarisation unveiling a unique function of lactate in driving cellular change. However, little is known whether lactylation can modify non-histone proteins. This led these researchers to question whether lactylation represents a widespread PTM that modifies non-histone proteins. Typically, identifying a PTM via mass spectrometry (MS) relies on the detection of a fixed mass shift on both the peptide precursor and tandem MS (MS/MS) fragment ions. Therefore, the research team sought to identify a reliable lactylation signature ion for MS analysis. Initially, they identified a cyclic immonium (CycIm) ion of lactyllysine (Klac) generated during MS/MS that accurately determined protein lactylation assignment. To show the validity and sensitivity of the CycIm ion as a marker of lactylation, they analysed the affinity-enriched lactyproteome of the HEK293T cells and nonlactylated spectral libraries. In addition, they demonstrated the ability of the CycIm ion to distinguish lactylated proteins in both human and cellular proteomes. Having confidently determined a lactylation diagnostic ion, they further showed the efficacy of the application of CycIm ion as a diagnostic ion-based strategy by screening the unenriched public human Meltome Atlas. Interestingly, they found that lactylation is a common PTM on glycolytic enzymes and a conserved PTM on fructose-bisphosphate aldolase A (ALDOA). By incorporating Klac, an unnatural amino acid, at the residue position 147 of human ALDOA, they showed lactylation has a site-specific inhibitory effect on ALDOA activity suggesting a lactylation-dependent feedback loop in glycolysis. Taken together, these researchers have shown that lactylation is a widespread PTM that is not restricted to histones and have developed a sensitive and validated method for lactylation identification by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Further investigation into the effects and targets of lactylation is necessary to unveil the biological importance of this PTM. 

How was PEAKS used?

Experimental and public proteomic data were searched using PEAKS Xpro against the Uniprot human proteome database. Parameters were set with a precursor mass tolerance of 20 ppm and fragment ion mass tolerance of 0.1 Da for the data collected on a SYNAPT G2-Si. Precursor mass tolerance was set to 10 ppm, and fragment ion mass tolerance was set to 0.02 Da for data acquired on an Oribitrap Eclipse Tribrid. For lactylation mining in public proteome data, the database search parameters were set as follows: for both the SILAC data acquired on a Q Exactive and the human tissue proteome data acquired on a LTQ Orbitrap Elite, the precursor mass tolerance was set to 10 ppm, and the fragment mass tolerance was set to 0.02 Da; for TMT MS2 quantification- based Meltome Atlas data, the precursor mass tolerance was set to 5 ppm, and the fragment mass tolerance was set to 0.02 Da. Additional parameters include: chymotrypsin (for recombinant proteins) or trypsin (cell lysate) as the enzyme, maximum of two missed cleavages and semi-specific digestion. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionine, acetylation of the N-terminal, and lactylation of lysine were set as variable modifications. Peptide-spectrum matches (PSM) were filtered at 1% FDR using a target-decoy strategy. For the determination of lactylation, the modified peptides had to reach an AScore ≥ 20, and then the corresponding MS/MS were subject to manual inspection.  When determining CycIm ion production from lactylated peptides, best PSMs for each identified peptide were assigned based on the -10lg P value and were selected as representatives for the analysis of CycIm ion production frequency and relative ion abundance. Relative ion abundances were calculated by the intensities of target ions normalised to the base peaks in the queried MS/MS spectra based on the MS/MS ion lists exported by PEAKS Studio.

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). doi.:10.1038/s41592-022-01523-1


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.