How to Detect Histone Lactylation Modifications Using High-Resolution Mass Spectrometry?
- DDA (Data-Dependent Acquisition): for exploratory studies
- DIA / PRM (Parallel Reaction Monitoring): for targeted quantitative analysis
- MS1 resolution ≥ 60,000
- MS2 resolution ≥ 30,000
- Dynamic exclusion: ≥30 s
- Ion source mode: positive ion (ESI+)
- Lactylation (Kla): +72.0211 Da
- Enable multiple variable modifications (e.g., acetylation, methylation) to assess specificity by comparison
- Use synthetic peptides to validate specificity
- Use Western blot / IP-MS to validate antibody specificity
- High-resolution Orbitrap MS platforms supporting multi-mode acquisition (DIA/PRM)
- High-coverage identification and quantitative analysis at the modification-site level
- Optimized enrichment workflows and stringent QC standards to ensure data stability
- Bioinformatics support, including pathway enrichment, regulatory network analysis, and cross-modification mapping
Histone lactylation is a newly identified post-translational modification, and its discovery has broadened our understanding of how metabolites participate in epigenetic regulation. Because histone lactylation typically occurs at low abundance and can be readily confounded with other modifications (e.g., acetylation) during mass-spectrometric interpretation, accurate detection as well as reliable qualitative and quantitative characterization generally require high-resolution mass spectrometry (HRMS).
Background: What Is Histone Lactylation?
Histone lactylation is a novel lysine modification first reported in 2019 by Zhang and colleagues in Nature, and it is derived from the lactate-related metabolite lactyl-CoA. This modification can influence chromatin structure and gene expression, and has been frequently implicated in physiological and pathological contexts such as hypoxia, inflammation, and tumor metabolic reprogramming.
Compared with common modifications such as acetylation, lactylation shows the following distinct features:
Why must the Detection of Lactylation Rely on High-Resolution Mass Spectrometry?
1. Several Challenges in Detecting Histone Lactylation
(1) Small mass differences between modifications
Lactylation (+72.0211 Da) differs from acetylation (+42.0106 Da) by 30 Da; with insufficient resolving power, spectra and peptide-spectrum matches can be misassigned.
(2) Complex modification-site landscape
Histones are enriched in lysine residues, and multiple candidate sites may be modified simultaneously; high resolving power is required to differentiate closely related species and confidently localize modification sites.
(3) Extremely low abundance
In endogenous biological samples, lactylation is typically far less abundant than acetylation/methylation and other common marks, necessitating a highly sensitive detection platform.
2. High-Resolution Mass Spectrometry Platforms (e.g., Orbitrap Fusion Lumos, timsTOF Pro 2)
(1) Ppm-level mass accuracy, enabling discrimination of lactylation from other chemically similar modifications
(2) High scan speed, supporting strategies such as data-independent acquisition (DIA) or PRM
(3) High sensitivity, which is advantageous for detecting low-abundance modified targets
At MtoZ Biolabs, we use an Orbitrap Exploris 480 coupled to a NanoLC system and develop dedicated enrichment and MS methods for low-abundance histone modifications to ensure data reproducibility and depth of coverage.
Detailed Workflow for Histone Lactylation Detection
1. Sample Preparation
(1) Sources: nuclear extracts, animal tissues, clinical samples, etc.
(2) Histone extraction: perform acid extraction (e.g., 0.4N H2SO4) to selectively enrich histones
(3) Reduction/alkylation: treat disulfide bonds
(4) Enzymatic digestion strategy: use combined proteases such as Trypsin/LysC to improve coverage of lactylated peptides
2. Enrichment of Modified Peptides
(1) Anti-lactylation antibody enrichment (immunoaffinity): commercially available anti-lactyl-lysine antibodies (e.g., PTMScan®) can be used for IP-grade enrichment
(2) Alternative methods: chemical derivatization labeling or HILIC-based fractionation strategies
3. LC-MS/MS Analysis
(1) Recommended platforms: Orbitrap Exploris, Fusion Lumos, QE HF, etc. can all be used.
(2) Acquisition modes:
(3) Key parameters:
4. Data Analysis
(1) Database search engines: MaxQuant, pFind, MSFragger, etc.
(2) Modification settings:
(3) Quantification strategies: LFQ, TMT, or PRM/Parallel Reaction Monitoring
(4) Result verification:
FAQs
Q1: Is lactylation modification easily misidentified as acetylation?
Yes. If instrument resolution is insufficient, or if the database search is not configured with the mass shift for lactylation, lactylated peptides may be incorrectly assigned as acetylated. Therefore, high resolving power and accurate mass matching are crucial.
Q2: Is enrichment necessary?
Enrichment is recommended. Although high-resolution instruments can detect low-abundance peptides, histone lactylation is very rare, and detection may be challenging without enrichment.
Q3: Can lactylation be analyzed using DIA?
Yes. DIA offers high throughput and enables retrospective re-interrogation, making it suitable for systematic studies. We recommend PRM or DIA for targeted quantification of lactylation modifications.
Advantage services of MtoZ Biolabs
At MtoZ Biolabs, leveraging advanced mass spectrometry platforms and extensive proteomics expertise, we provide one-stop histone lactylation detection services:
Histone lactylation is increasingly recognized as a major topic in epigenetics, serving as a mechanistic link between metabolic state and chromatin structure and being particularly relevant to studies of tumor immunity, chronic inflammation, and stem cell fate. With high-resolution mass spectrometry, researchers can more precisely interrogate this subtle yet functionally important modification. If you are conducting related research, you are welcome to contact MtoZ Biolabs. We will tailor a lactylation detection plan to support your scientific studies.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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