How to Determine Lactylation Sites
Lactylation is a newly identified post-translational modification in which lactate is covalently linked to lysine residues in proteins. This modification plays a critical role in cellular metabolism, particularly in lactate metabolism and immune responses. Lactylation sites are defined as the specific lysine residues within proteins where this modification occurs.
Mass spectrometry represents the gold standard for the identification of lactylation sites in proteins. Through the separation and analysis of proteins or peptide fragments, the precise amino acid residues undergoing lactylation can be accurately determined. The general workflow involves the following steps:
1. Sample Preparation
Select appropriate cell or tissue samples with robust lactate metabolic activity to ensure sufficient levels of potential lactylation.
2. Protein Extraction and Digestion
Isolate proteins from the selected samples and enzymatically digest them into peptides.
3. Mass Spectrometry Analysis
Perform liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze the peptide mixtures and detect lactylated peptides.
4. Data Processing and Analysis
Utilize dedicated bioinformatics tools to process LC-MS/MS data, identify lactylated peptides, and localize the modification sites. This typically includes database searching, peptide-spectrum matching, and modification site assignment.
5. Bioinformatics Analysis
Conduct further analysis of identified lactylation sites, including functional annotation, structural characterization, and comparative analysis with other known post-translational modifications.
6. Experimental Validation
Validate key findings using techniques such as Western blotting with antibodies specific to lactylation.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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