Workflow of Protein Lactylation Analysis

Protein lactylation is an emerging post-translational modification (PTM) that plays a significant role in various biological processes. As research progresses, the potential functions of lactylation in metabolic regulation, immune responses, and disease pathogenesis are gradually being uncovered. To explore the functional implications of protein lactylation, scientists have developed a precise and efficient analytical workflow.

 

Sample Preparation

Sample preparation is the critical first step in lactylation analysis. Samples can be derived from various biological sources such as cell lines, tissues, or bodily fluids. Once collected, the samples need to be lysed to release proteins. This is typically achieved using sonication or repeated freeze-thaw cycles in the presence of lysis buffer. Protease inhibitors must be added during lysis to prevent protein degradation.

 

Protein Extraction and Quantification

Following lysis, the next step is to extract and quantify the proteins. Common methods for protein extraction include precipitation (e.g., ethanol precipitation) and centrifugation. Protein quantification is usually performed using the BCA or Bradford assay, which helps to ensure consistency in sample concentration for subsequent analyses.

 

Protein Digestion

To detect lactylation modifications, protein samples are typically digested into smaller peptides. The goal of digestion is to break down the proteins into peptides, making them more accessible for identification in mass spectrometry analysis. The most commonly used enzyme is trypsin, which specifically cleaves at the carboxyl side of lysine and arginine residues. Digestion is generally carried out at 37°C for 12 to 16 hours.

 

Peptide Enrichment

Due to the typically low abundance of lactylation modifications in proteins, direct detection may be hindered by non-specific background signals. Therefore, prior to mass spectrometry analysis, it is necessary to enrich the lactylated peptides. This is commonly done using specific antibodies against lactylation modifications through immunoprecipitation (IP), allowing for the enrichment of target peptides from a complex peptide mixture.

 

Mass Spectrometry Analysis

Mass spectrometry (MS) is the central technique for detecting and quantifying lactylation modifications. By using liquid chromatography-tandem mass spectrometry (LC-MS/MS), peptides can be separated and analyzed by mass spectrometry. The MS data provides precise information on the mass of the peptides, as well as the location and abundance of the lactylation modification. Data analysis software, such as MaxQuant or Proteome Discoverer, is used to further process the MS data, identifying and quantifying lactylated peptides.

 

Data Analysis and Interpretation

The data generated from mass spectrometry analysis is often complex and voluminous, requiring bioinformatics tools for processing. By aligning peptide sequences and identifying modification sites, researchers can generate a comprehensive overview of lactylation modifications. The results of data analysis help researchers understand the role of lactylation in biological processes, revealing its potential biological significance.

 

Validation and Functional Studies

After identifying potential lactylation sites, further validation and functional studies are essential. This is typically done through point mutation analysis, Western blotting, or immunofluorescence staining to confirm the presence of lactylation modifications and their role in biological processes.

 

The workflow for protein lactylation modification analysis provides a powerful tool for in-depth research into this emerging post-translational modification. Each step, from sample preparation to data analysis, requires precision and stringent control to ensure the accuracy and reliability of the results.