Meeting the Challenge of Lactylation Analysis
Lysine lactylation (Kla) was first described in 2019 as a histone mark derived from the glycolytic metabolite L-lactate, establishing a direct molecular link between cellular metabolism and epigenetic regulation. Unlike many PTMs with dedicated writer-enzyme systems, lactylation is driven by non-enzymatic and enzymatic transfer of lactyl groups from lactyl-CoA, with the cellular lactate pool — modulated by glycolysis, hypoxia, and mitochondrial function — directly shaping the Kla landscape. This metabolic dependency makes lactylation uniquely positioned as a sensor of cellular energy status and a mediator of metabolic memory in physiological and pathological contexts.
Why Lactylation Demands a Dedicated Analytical Approach
Lactylation presents distinct analytical challenges that differentiate it from other lysine acylations. Kla peptides carry a mass shift of +72.021 Da from the lactyl group (C₃H₄O₂), significantly larger than acetylation (+42.011 Da) but distinct from other acyl-lysine modifications including propionylation (+56.026 Da), butyrylation (+70.042 Da), crotonylation (+68.026 Da), and succinylation (+100.016 Da). Reliable discrimination of Kla from these structurally similar modifications requires high-resolution mass spectrometry with accurate mass measurement, combined with site-specific fragmentation evidence and database search strategies that account for the full acylation landscape. Our platform addresses these challenges with optimized antibody enrichment validated for Kla specificity, high-resolution Orbitrap acquisition for unambiguous mass assignment, and multi-engine search strategies for confident Kla site identification. For broader context on lysine acylation analysis, our related Acylation Quantitative Proteomics service provides multi-acylation profiling capabilities across modification types.
Our lactylation platform integrates multiple analytical strategies — from global Kla discovery in metabolic and disease models to targeted quantification of specific lactylation sites — allowing researchers to select the analytical depth that matches their biological question. Whether mapping the lactylproteome of a tumor model under metabolic stress, profiling histone Kla dynamics in response to glycolytic modulation, or quantifying specific Kla sites in cardiac tissue after ischemia-reperfusion injury, our services deliver the site-resolved data needed for confident biological interpretation. For integration with broader PTM discovery efforts, our Pan PTM Proteomics service provides multi-modification profiling workflows.
Our Lactylation Analysis Service Portfolio
We offer a structured portfolio of lactylation analysis services designed to address specific research objectives — from global Kla site discovery to targeted quantification of individual lactylation events. The table below maps common research goals to our recommended service modules.
| Research Objective |
Recommended Service |
Key Technology |
| Global Kla site identification and lactylproteome discovery |
Global Kla Profiling |
Anti-Kla antibody enrichment, high-resolution Orbitrap MS, MaxQuant/PD search |
| Quantitative comparison of lactylation across conditions |
Quantitative Lactylproteome Analysis |
Label-free quantification, TMT labeling, SILAC, PRM validation |
| Site-specific lactylation validation and targeted quantification |
Targeted Kla Site Verification |
PRM-based targeted MS with synthetic Kla peptide standards |
| Integrated lactylation-acetylation crosstalk analysis |
Kla-Kac Dual Profiling |
Parallel anti-Kla and anti-Kac enrichment, dual-modality LC-MS/MS |
Each service module is available independently or can be combined into an integrated lactylation characterization workflow. For studies investigating the interplay between lactylation and other lysine acylations, our PTM Crosstalk Analysis service provides multi-modification co-regulation insights.
Integrated Technical Platform for Lactylation Proteomics
Reliable lactylation analysis depends on optimized methods across the entire analytical pipeline — from antibody-based enrichment through high-resolution mass spectrometry acquisition to computational Kla site localization. Our platform integrates best-in-class approaches at each stage, configurable to match the complexity and objectives of your project.
Anti-Lactyl-Lysine Antibody Enrichment
Lactylated peptides constitute a minor fraction of the total peptidome and require specific enrichment for reliable LC-MS/MS detection. We deploy anti-lactyl-lysine (anti-Kla) antibody-based immunoaffinity enrichment as the primary strategy, using commercially sourced and internally validated pan-Kla antibodies that recognize lactylated lysine residues irrespective of surrounding sequence context. Antibody specificity is verified through competition assays with acetylated, butyrylated, and crotonylated peptide standards to confirm selective Kla recognition. For projects requiring comprehensive Kla coverage, we employ serial enrichment strategies that maximize the depth of the detected lactylproteome. Enrichment efficiency is monitored through on-bead quality control steps including LC-MS/MS analysis of bound and flow-through fractions.
High-Resolution LC-MS/MS Acquisition
Kla peptide analysis is performed on high-resolution Orbitrap platforms, optimized for the distinct mass shift and fragmentation characteristics of lactylated peptides. The +72.021 Da mass shift is resolved from other lysine acylations through high-resolution precursor ion measurement (≥60,000 resolution at m/z 200). Nano-flow LC configurations maximize sensitivity for limited sample amounts, while capillary-flow systems provide throughput for larger cohort studies. Data-dependent acquisition with dynamic exclusion is the standard for discovery lactylproteomics, with scheduled PRM methods developed for targeted Kla site quantification. Sequential fractionation strategies (high-pH RP, basic RP) are employed for deep lactylproteome coverage in projects requiring comprehensive site identification.
Multi-Engine Database Search and Kla Site Localization
Kla site identification presents computational challenges due to the combinatorial search space of variable modifications. We deploy a multi-engine search strategy using MaxQuant with Kla (+72.021 Da on lysine) and oxidation of methionine as variable modifications, Proteome Discoverer with the PTM workflow for cross-modality identification, and pGlyco/pFind for open-search approaches that detect unexpected modifications including co-occurring acylations. Kla site localization confidence is validated against key quality criteria — Andromeda score or equivalent, localization probability, and mass accuracy tolerance — with ambiguous assignments resolved through manual spectral inspection. Multi-engine cross-validation maximizes identification confidence while minimizing false positives from the expanded search space.
Quantitative Lactylproteome Strategies
Lactylation is dynamically regulated by metabolic flux, making quantitative information essential for biological interpretation. Our platform supports label-free quantification using extracted ion chromatogram alignment for discovery studies and pilot experiments, TMT labeling for multiplexed comparisons of up to 16 conditions within a single experiment, and PRM-based targeted quantification with synthetic Kla peptide standards for site-specific validation. Differential analysis covers both global Kla abundance changes and site-specific lactylation dynamics.
Lactylation Proteomics Workflow: From Sample to Publication-Ready Data
Step 1: Sample Preparation and Anti-Kla Enrichment
Proteins are extracted, reduced, alkylated, and digested using optimized proteases (trypsin or Lys-C/trypsin combinations). Kla-peptides are enriched using validated anti-lactyl-lysine antibody-conjugated beads. Enriched peptides are desalted and fractionated as needed for deep coverage projects. Input controls are retained for normalization.
Step 2: LC-MS/MS Data Acquisition
Kla-enriched peptides are analyzed on high-resolution Orbitrap platforms with optimized LC gradients and data-dependent acquisition. High-resolution precursor scans (≥60,000) discriminate Kla from other acylations by accurate mass. Stepped HCD fragmentation provides sequence-informative b- and y-ions for site localization.
Step 3: Kla Peptide Identification and Site Localization
Raw MS data are searched using MaxQuant, Proteome Discoverer, and open-search engines with Kla (+72.021 Da) as a variable modification. Site localization is scored using MaxQuant localization probability and validated against mass accuracy, spectral quality, and fragment ion coverage criteria. Cross-validation across search engines maximizes confidence.
Step 4: Quantitative Analysis and Differential Kla Profiling
Kla peptide abundance is quantified using label-free (XIC alignment), TMT (reporter ion intensities), or SILAC (heavy/light ratios) approaches depending on experimental design. Differential expression analysis identifies significantly regulated Kla sites across conditions with appropriate statistical testing and multiple hypothesis correction.
Step 5: Motif Analysis and Functional Annotation
Kla site sequence context is analyzed for enriched motifs using plogo and MEME-based approaches. Functional annotation includes Gene Ontology enrichment, KEGG pathway mapping, protein-protein interaction networks, and integration with published Kla datasets. For projects with matched proteome data, Kla site regulation is evaluated independently of protein abundance changes.
Step 6: Deliverables and Scientist Review
Complete lactylation dataset including Kla site identification table with localization confidence scores, quantitative data with statistical analysis, annotated MS/MS spectra for identified Kla sites, motif analysis and pathway enrichment results, and a scientist consultation session for biological interpretation of lactylation findings in the context of your metabolic or disease model.
For unbiased discovery of additional co-occurring modifications beyond lactylation, our Open-Search PTM Discovery service provides comprehensive multi-modality identification capabilities.
Lactylation Proteomics in Biomedical and Metabolic Research
Lactylation research spans a rapidly expanding range of biological contexts, reflecting the fundamental role of this modification as a metabolic-epigenetic signaling hub. Our platform is configured to support the specific requirements of each application domain, from exploratory Kla discovery in metabolic disease models to targeted analysis in cancer and cardiovascular research.
Cancer Metabolism and Tumor Microenvironment
The Warburg effect — aerobic glycolysis in cancer cells — drives local lactate accumulation within the tumor microenvironment, creating conditions conducive to elevated protein lactylation. Kla on histones directly activates transcription of genes involved in cell proliferation, migration, and metabolic reprogramming, positioning lactylation as a key epigenetic mediator of the glycolytic phenotype. Our global Kla profiling workflow is optimized for tumor tissue, cancer cell lines under metabolic stress, and tumor interstitial fluid, delivering comprehensive site-specific Kla maps that capture the lactylation landscape associated with cancer progression and therapy resistance.
Cardiovascular and Ischemia-Reperfusion Injury
Lactate levels rise dramatically in ischemic tissues, driving acute changes in protein lactylation that can have either protective or pathological consequences depending on cellular context. Recent studies have demonstrated that Kla of specific proteins — including serpin family members, heat shock proteins, and metabolic enzymes — modulates apoptosis, inflammation, and tissue remodeling in the heart and brain following ischemic injury. Our platform supports quantitative Kla profiling in cardiac and neural tissues, enabling identification of therapeutically relevant lactylation events.
Immunometabolism and Inflammation
Immune cell activation is accompanied by profound metabolic reprogramming — including the switch to aerobic glycolysis (similar to the Warburg effect) — that directly shapes the Kla landscape in immune cells. Lactylation of histones and immune signaling proteins has been implicated in macrophage polarization, T cell differentiation, and inflammatory gene regulation. Our lactylation analysis services enable investigation of how metabolic-immune crosstalk is mediated through protein Kla at the molecular level.
For comprehensive quantitative analysis of lactylation dynamics across large sample sets, our PTM Quantitative Analysis Services platform provides integrated multiplexed quantification workflows.
Case Study: Lactyloproteomic Profiling Reveals Serpina3k Lactylation Protects Against Cardiac Ischemia-Reperfusion Injury
A 2025 study by Wang et al. published in Nature Communications applied global lactylome and proteome profiling combined with functional validation to discover that lactylation of Serpina3k at lysine 351 (K351la) protects the heart from ischemia-reperfusion (I/R) injury through a paracrine signaling mechanism, demonstrating the power of integrated lactyloproteomic analysis to uncover functionally significant lactylation events in disease-relevant models.
Background: Myocardial ischemia-reperfusion injury remains a major clinical challenge in cardiac surgery and interventional cardiology. While lactate — the source of protein lactylation — accumulates dramatically in ischemic myocardium, the role of protein lactylation in cardiac I/R pathophysiology was entirely unknown before this study. The authors hypothesized that the I/R-induced surge in lactate production drives acute remodeling of the cardiac lactylome, with specific Kla events exerting functional effects on cell survival and tissue repair.
Approach: The team established a mouse model of myocardial I/R injury and performed integrated 4D label-free lactylome and proteome profiling of infarct zone tissue using anti-Kla antibody enrichment followed by high-resolution LC-MS/MS. Bioinformatics analysis included differential Kla site quantification, motif analysis, protein-protein interaction networks, and integration of lactylome with proteome data to distinguish site-specific regulation from protein abundance changes. Functional validation through site-directed mutagenesis, recombinant protein studies, and cellular assays confirmed the mechanistic role of Serpina3k K351 lactylation.
Key Findings:
- A total of 1,674 Kla sites were identified on 380 proteins from mouse myocardium, with 1,472 sites quantified, representing the first comprehensive lactylome atlas of cardiac I/R injury
- Ischemia-reperfusion induced robust Kla upregulation at 71 sites (versus only 25 in myocardial infarction alone), revealing I/R-specific lactylation signatures distinct from ischemic remodeling
- Serpina3k (SA3K) — a member of the serine protease inhibitor family — emerged as one of the most strongly Kla-upregulated proteins in I/R, with lactylation at K351 enhancing SA3K protein stability through reduced ubiquitin-proteasome degradation
- Cardiac fibroblasts secrete lactylated SA3K, which protects cardiomyocytes from I/R-induced apoptosis in a paracrine manner through inhibition of the WNT pathway and activation of RISK and SAFE survival signaling cascades
- Gene Ontology analysis of differentially lactylated proteins revealed enrichment in biological processes including wound healing, response to hypoxia, protein folding, and metabolic process regulation, identifying lactylation-responsive pathways in the injured myocardium
Significance: This study demonstrates that integrated lactyloproteomic profiling — combining global Kla site discovery with quantitative analysis and functional validation — can uncover therapeutically relevant lactylation events in disease models. The finding that Serpina3k lactylation mediates cardioprotective signaling provides a molecular entry point for understanding how metabolic stress signals are communicated through Kla and opens new directions for targeting lactylation in cardiac injury. For researchers investigating lactylation in any disease context, our proteomics platform provides the analytical depth to map Kla sites, identify regulated pathways, and prioritize functional candidates.
Figure 1 from Wang et al. (2025). Global lactylome profiling of myocardial ischemia/reperfusion injury. (a) Experimental design — sham, MI, and I/R mouse groups with 4D label-free lactylome and proteome analysis. (b–c) Kla site and protein density distributions showing differential regulation in MI and I/R. (d–e) Numbers of significantly changed Kla sites and proteins. (f,g) Heat maps and GO enrichment of differentially lactylated sites. (h,i) Heat maps and GO enrichment of differentially expressed proteins. (j) STRING protein-protein interaction network of lactylated proteins in I/R. (CC BY 4.0)
Representative Lactylation Proteomics Data Outputs
Our lactylation analysis pipeline delivers multi-dimensional data outputs that provide a complete picture of the lysine lactylation landscape at site-specific resolution. Below are representative examples of the key data types included in every project deliverable. These outputs are generated for every identified Kla site, enabling detailed assessment of lactylation identity, site localization confidence, and quantitative changes across experimental conditions.
Representative lactylation proteomics data. (Left) Kla site identification table with protein ID, site position, localization probability, sequence motif, and abundance ratios. (Center) Quantitative comparison of Kla levels across conditions — significant sites colored by regulation direction. (Right) Annotated MS/MS spectrum showing diagnostic fragment ions confirming Kla site localization with the characteristic +72.021 Da mass shift.
Every data deliverable is reviewed by our lactylation proteomics scientists, who verify spectral quality, confirm Kla site assignments, and provide biological context for the results in the context of metabolic and epigenetic signaling. Custom visualization and data formatting options are available to match publication requirements or internal reporting standards.
Why Choose Our Lactylation Proteomics Analysis Services
Validated Kla-Specific Enrichment
Our anti-lactyl-lysine antibody enrichment platform is verified through competition assays with acetylated, butyrylated, and crotonylated peptide standards, ensuring selective Kla recognition without cross-reactivity. Enrichment efficiency is monitored at every stage through on-bead quality controls, providing confidence that identified Kla sites represent genuine lactylation events.
High-Resolution Acylation Discrimination
Lysine acylation modifications share overlapping mass shifts that challenge confident identification. Our Orbitrap-based acquisition at ≥60,000 resolution, combined with accurate mass measurement and multi-engine database searching, provides unambiguous discrimination of Kla from other acyl-lysine modifications including acetylation, propionylation, butyrylation, crotonylation, and succinylation.
Comprehensive Quantitative Strategies
We offer the full spectrum of quantification approaches — label-free, TMT, SILAC, and PRM — from discovery Kla profiling to targeted site validation. Each strategy is optimized for the specific experimental design and biological question, ensuring appropriate statistical power to detect regulated Kla sites even when lactylation changes are modest in magnitude.
Cross-Modality Bioinformatics Integration
Lactylation rarely acts in isolation — it competes with and cross-regulates other lysine acylations at shared sites. Our bioinformatics pipeline integrates Kla data with acetylation (Kac), crotonylation (Kcr), and other acylation datasets when available, enabling systems-level analysis of the lysine acylation landscape and identification of modification cross-talk at individual residue positions.
Related Services
Our lactylation analysis services are supported by a broader PTM characterization platform offering complementary analytical capabilities. These services can be combined to address multi-dimensional PTM characterization needs across lysine acylation and other modification types.
- Global PTM Profiling — Broad multi-PTM discovery analysis encompassing lactylation alongside other lysine acylations and PTM classes
- Crotonylation Analysis — Targeted lysine crotonylation (Kcr) profiling for comparative acylation studies alongside lactylation analysis
- Succinylation Analysis — Comprehensive lysine succinylation (Ksu) identification and quantification for multi-acylation landscape mapping
- Malonylation Proteomics — Specialized lysine malonylation (Kma) analysis for expanded acylation modification profiling
- MS-Based PTM Analysis — Comprehensive framework for multi-modification discovery across diverse PTM types and sample types
- PTM Proteoform Mapping — Detailed characterization of combinatorial PTM patterns on individual protein proteoforms including lactylation co-occurrence
- PTM Bioinformatics Analysis — Advanced bioinformatics for PTM data integration, functional annotation, and cross-modification analysis
Frequently Asked Questions
What is lysine lactylation (Kla) and why is it biologically significant?
Lysine lactylation (Kla) is a post-translational modification in which a lactyl group derived from the glycolytic metabolite L-lactate is covalently attached to lysine residues. First discovered on histones in 2019, Kla directly links cellular metabolic state to gene regulation — elevated lactate production (e.g., during the Warburg effect in cancer, hypoxia, or immune activation) drives increased histone and non-histone protein lactylation, which in turn regulates transcription, protein function, and cell behavior. Kla is now recognized as a central mechanism of metabolic-epigenetic communication with implications in cancer, cardiovascular disease, immunology, and neurodegeneration.
How do you specifically enrich lactylated peptides?
We use anti-lactyl-lysine (anti-Kla) antibody-based immunoaffinity enrichment as our primary strategy. Pan-Kla antibodies are validated through competition assays with acetylated, butyrylated, and crotonylated peptide standards to confirm selective Kla recognition. Enriched Kla peptides are analyzed by high-resolution LC-MS/MS, and enrichment specificity is monitored through on-bead quality control steps. For deep Kla coverage projects, we employ serial enrichment or pre-fractionation strategies to maximize the depth of the detected lactylproteome.
How do you distinguish lactylation from other lysine acylations?
Kla (+72.021 Da) is distinguished from other lysine acylations through a combination of high-resolution precursor ion measurement (≥60,000 resolution at m/z 200) for accurate mass assignment, retention time characteristics, diagnostic fragment ions, and multi-engine database searching that includes Kla and other acyl modifications as variable modifications. The distinct mass shift and fragmentation signature of Kla allow confident discrimination from acetylation (+42.011 Da), propionylation (+56.026 Da), butyrylation (+70.042 Da), crotonylation (+68.026 Da), and succinylation (+100.016 Da).
What sample types are compatible with your lactylation analysis workflow?
Our pipeline accepts a wide range of sample types including cultured cells (≥1×10⁷ cells for global Kla profiling), tissue samples (≥20 mg, with particular experience in cardiac, liver, brain, and tumor tissues), and biofluids with demonstrated lactylation signals. For cell culture experiments, we recommend lactate treatment or metabolic modulation to enhance the detectable Kla signal when appropriate. Samples should be collected with metabolic quenching to preserve the endogenous lactylation state.
What quantification strategies are available for lactylation analysis?
We offer label-free quantification using extracted ion chromatogram alignment for discovery Kla profiling, TMT labeling for multiplexed comparison of up to 16 conditions, SILAC for metabolic labeling in cell culture models, and PRM methods with synthetic Kla peptide standards for targeted absolute quantification. For studies requiring integration of Kla data with matched proteome abundance, we recommend combining lactylome profiling with parallel proteome analysis to distinguish site-specific regulation from protein-level changes.
Can you analyze lactylation in combination with other lysine acylations?
Yes — our platform supports parallel enrichment and analysis of lactylation alongside acetylation, crotonylation, succinylation, and other lysine acylations from the same biological sample. This dual- or multi-modality approach enables comprehensive mapping of the lysine acylation landscape and identification of modification cross-talk at individual residue positions where different acyl groups may compete for the same lysine. Parallel quantification across modification types provides a complete picture of how metabolic flux shapes the cellular acylation state.
How does your bioinformatics analysis support lactylation data interpretation?
Our bioinformatics pipeline extends beyond standard identification workflows to include Kla-specific motif analysis using plogo and MEME approaches, functional enrichment analysis (GO, KEGG, Reactome) of lactylated proteins, protein-protein interaction network mapping, integration with published Kla datasets from the literature, and cross-modality analysis when parallel acylation data is available. Results are delivered with interactive visualization and a scientist consultation session that provides biological context for Kla findings within your specific metabolic or disease model.
References
- Wang L, Li D, Yao F, Feng S, Tong C, Rao R, Zhong M, Wang X, Feng W, Hu Z, Jin B, Wang L, Hu S, Zhou B. Serpina3k lactylation protects from cardiac ischemia reperfusion injury. Nat Commun. 2025;16:1012.
- He J, Lai T, Zhou Z, Yang H, Lei Z, Zhou L, Li N, He Y, Zeng S, Munai E, Tan Y, Wang M, Zhang Y, Zhou W, Wu Y. Multiomics profiling reveals the involvement of protein lactylation in nonhomologous end joining pathway conferring radioresistance in lung adenocarcinoma cell. Sci Rep. 2025;15:24651.
- Niu Z, Chen C, Wang S, Lu C, Wu Z, Wang A, Mo J, Zhang J, Han Y, Yuan Y, Zhang Y, Zang Y, He C, Bai X, Tian S, Zhai G, Wu X, Zhang K. HBO1 catalyzes lysine lactylation and mediates histone H3K9la to regulate gene transcription. Nat Commun. 2024;15:3561.
For research use only. Not for use in diagnostic procedures.
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