Step-by-Step Workflow for Quantitative Analysis of Protein Lactylation

Protein lactylation is an emerging lysine post-translational modification that has recently gained significant attention in epigenetics, immunometabolism, and cancer biology. In contrast to well-established modifications such as acetylation and phosphorylation, analytical strategies for the detection and quantitative characterization of lactylation remain under active development. Consequently, researchers frequently encounter challenges related to experimental design, sample preparation, and data interpretation. This article provides a systematic overview of the standard workflow for quantitative analysis of protein lactylation, covering all critical steps from sample preparation to mass spectrometry-based data analysis, with the aim of facilitating the acquisition of high-quality and reproducible lactylation proteomics datasets.

Analytical Challenges in Quantitative Protein Lactylation Studies

Protein lactylation presents several inherent analytical challenges:

1. Limited Knowledge of Modification Sites

To date, only several hundred lactylation sites have been reported, and current database coverage remains incomplete.

 

2. Low Modification Abundance and Weak Signals

Lactylation typically occurs on low-abundance proteins or at a low stoichiometry on lysine residues.

  

3. Scarcity of Highly Specific Antibodies

Commercially available anti-lactylation antibodies are limited, and their specificity requires careful validation.

  

4. Substantial Interference from Co-Existing Modifications

Lactylation sites often overlap with acetylation, formylation, and other lysine modifications, complicating confident discrimination.

As a result, high-quality lactylation proteomics studies depend on precise enrichment strategies, highly sensitive mass spectrometry platforms, and robust high-throughput data analysis pipelines.

 

Workflow for Quantitative Analysis of Protein Lactylation

The overall analytical workflow consists of six core steps:

1. Sample Pretreatment and Protein Extraction

Appropriate selection of sample types and processing conditions represents the first critical determinant of experimental success:

(1) Sample Types: Cells, tissues, serum, or other biological fluids can be analyzed, with particular attention to lactate metabolic activity.

(2) Protein Extraction Buffers: Lysis buffers containing detergents and enzymatic inhibitors are recommended to preserve endogenous modification states.

(3) Protein Quantification and Quality Control: Consistent protein input across samples is essential to minimize systematic bias.

It is recommended to supplement lysis buffers with histone deacetylase and de-lactylase inhibitors (e.g., NAM and TSA) to protect endogenous lactylation modifications.

2. Proteolytic Digestion and Peptide Preparation

(1) Proteins are typically digested using trypsin.

(2) Combined digestion with Lys-C can further enhance proteolytic efficiency.

(3) Digested peptides should be desalted and purified using C18 columns to remove interfering substances.

High-quality enzymatic digestion is a prerequisite for reliable identification of lactylated peptides. Peptide-level quality control (e.g., BCA assays or SDS-PAGE analysis) is recommended to assess digestion completeness.

3. Enrichment of Lactylated Peptides

Strategy 1: Immunoaffinity Enrichment Using Anti-Lactylation Antibodies

• Currently, immunoaffinity enrichment with anti-Kla-specific antibodies represents the most widely adopted approach.

• Crosslinked protein A/G magnetic beads are recommended to reduce heavy-chain contamination.

• Multiple rounds of immunoprecipitation or sequential enrichment can be employed to improve capture efficiency.

Strategy 2: Chemical Derivatization Combined with Affinity Purification

• Selective derivatization based on the chemical properties of lactyl groups.

• Subsequent affinity-based enrichment of lactylated peptides.

• This approach is particularly suitable when antibodies are unavailable or for studies focusing on non-histone lactylation.

MtoZ Biolabs has optimized immunoprecipitation (IP) conditions and buffer systems for protein lactylation, thereby enhancing the specificity and reproducibility of lactylated peptide enrichment.

4. LC-MS/MS Analysis

(1) Instrument Platforms

• High-resolution mass spectrometers such as Orbitrap Fusion Lumos or Exploris 480 are recommended.

• These instruments are typically coupled with nano-flow liquid chromatography (nanoLC) systems to enhance analytical sensitivity.

(2) Acquisition Strategies

• DDA (Data Dependent Acquisition) is suitable for comprehensive discovery of lactylation sites.

• DIA (Data Independent Acquisition) enables quantitative analysis of predefined modification sites.

• PRM (Parallel Reaction Monitoring) is applied for targeted validation of specific lactylation events.

Leveraging extensive expertise in post-translational modification (PTM) analysis, MtoZ Biolabs has developed lactylation-specific mass spectrometry database search strategies that enable reliable identification of low-abundance lactylated peptides.

5. Database Searching and Modification Site Assignment

Software platforms such as MaxQuant, Proteome Discoverer, or Spectronaut can be employed using the following parameters:

• Modification Type: Lysine lactylation (+72.0211 Da).

• Protease Specificity: Trypsin/P.

• Databases: UniProt or RefSeq human/mouse protein databases.

• Mass Tolerance: precursor ions ±10 ppm; fragment ions ±0.02 Da.

• A false discovery rate (FDR) threshold of 1% should be applied, and high-confidence lactylation sites should be selected based on peptide scores and site localization metrics (e.g., PTM scores).

6. Quantitative Analysis and Bioinformatics Interpretation

(1) Quantification Approaches

• Label-based strategies (e.g., TMT/iTRAQ) enable multiplexed analysis of multiple sample groups and are well suited for dynamic lactylation studies.

• Label-free approaches do not require chemical labeling and are appropriate for exploratory analyses involving large sample cohorts.

(2) Downstream Bioinformatics Analyses

• GO and KEGG pathway enrichment analyses to elucidate functional networks associated with lactylated proteins.

• Motif analysis to identify sequence preferences surrounding modification sites.

• Co-modification mapping to investigate regulatory crosstalk with acetylation and phosphorylation.

MtoZ Biolabs offers integrated end-to-end services spanning raw mass spectrometry data processing and downstream biological function interpretation, thereby facilitating a comprehensive understanding of the biological significance of protein lactylation modifications.

The discovery of protein lactylation has provided novel insights into the mechanistic links between cellular metabolism and gene expression regulation. Although the analytical detection of this modification remains technically challenging, the integration of high-sensitivity mass spectrometry platforms, highly specific enrichment strategies, and well-established bioinformatics pipelines has enabled researchers to progressively elucidate its critical roles across diverse biological processes. MtoZ Biolabs offers comprehensive end-to-end quantitative analysis services for protein lactylation, encompassing protein sample preparation and modification site enrichment, high-resolution LC-MS/MS analysis and data interpretation, as well as customized quantitative strategy design (including TMT, DIA, PRM, and related approaches). We welcome collaboration and technical consultation to provide tailored experimental solutions or one-on-one project assessments, supporting robust and reliable advancement in lactylation research.

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

Related Services

Protein Lactylation Modification Analysis Service