How Label-Free Quantification Facilitates Precise Profiling of Lactylated Proteins
In recent years, lactylation has emerged as a novel post-translational modification (PTM) with crucial roles in biological processes such as metabolic regulation, inflammatory response, and tumorigenesis. However, due to its low abundance, diverse modification sites, and the absence of highly specific antibodies, research on lactylated proteins faces considerable technical challenges. To enable accurate detection and quantification of these proteins, label-free quantification (LFQ) has become a preferred and increasingly mainstream approach.
Lactylation: A Novel Link Between Metabolism and Epigenetics
Lactylation is a lysine-targeted post-translational modification originating from the intracellular accumulation of lactate. First reported in 2019, its discovery revealed that metabolic intermediates can directly influence gene expression, thereby bridging the fields of metabolism and epigenetic regulation. Emerging studies have demonstrated that lactylation contributes to processes such as macrophage-mediated inflammatory responses, tumor metabolic reprogramming, and stem cell differentiation. For instance, lactate-induced histone lactylation has been shown to enhance the expression of pro-inflammatory genes, indicating a potential regulatory role in the tumor immune microenvironment.
Why Employ Label-Free Quantification for Studying Lactylated Proteins?
Compared to isotope- or chemically-labeled quantification strategies such as TMT and iTRAQ, label-free quantification offers distinct advantages when investigating low-abundance PTMs like lactylation:
1. Minimizes Sample Handling Complexity ： Ideal for Precious Samples
Lactylation typically occurs under specialized physiological conditions such as cellular stress or hypoxia, where sample availability is inherently limited. Unlike labeling techniques, LFQ eliminates the need for stable isotope or chemical labeling, thereby reducing sample loss and technical variability. This makes it particularly suitable for rare tissue samples or small-scale animal model studies.
2. Greater Throughput and Proteome Coverage
Leveraging high-resolution mass spectrometry platforms (e.g., Orbitrap Exploris 480), LFQ enables comprehensive profiling and accurate quantification of tens of thousands of peptides. This deep coverage is essential for characterizing the lactylated proteome and elucidating the dynamic nature of lactylation under different biological conditions.
3. Seamless Integration with Enrichment Strategies to Enhance Sensitivity
Given the inherently low abundance of lactylation, targeted enrichment using specific antibodies or affinity-based purification is essential. LFQ can be flexibly integrated with proteomic workflows before and after enrichment, enabling reliable identification of modification sites and accurate quantification of site-specific expression changes.
Optimized Experimental Workflow: The Key to Accurate Capture of Lactylated Proteins
At MtoZ Biolabs, we have developed an LFQ-based workflow optimized for studying low-abundance PTMs such as lactylation. The following outlines our core experimental strategies:
1. Sample Lysis and Protein Extraction
Proteins are extracted using an optimized lysis buffer supplemented with phosphatase, deacetylase, and delactylase inhibitors to preserve the integrity of PTM states during sample processing.
2. Protein Digestion and Peptide Purification
Samples undergo enzymatic digestion with Trypsin or Lys-C, followed by C18 solid-phase extraction to remove salts and other contaminants, ensuring high-purity peptide mixtures.
3. Enrichment of Lactylated Peptides
Immunoaffinity enrichment is performed using commercially available anti-lactyllysine (Anti-Kla) antibodies to selectively isolate lactylated peptides, thereby increasing detection sensitivity.
4. LC-MS/MS-Based Label-Free Quantification
Quantitative analysis is conducted using high-resolution Orbitrap mass spectrometry, employing either data-dependent (DDA) or data-independent acquisition (DIA) modes. Software such as MaxQuant and Spectronaut is used for quantitative calculations and statistical evaluation.
5. Data Mining and Functional Annotation
Bioinformatics tools including GO, KEGG, and protein-protein interaction (PPI) networks, are employed to interpret the biological significance of lactylated proteins, uncovering enriched pathways and key regulatory hubs.
MtoZ Biolabs: Comprehensive Services for Lactylated Proteomics
1. End-to-End Solution for Lactylated Proteomics
From sample preparation to data interpretation, our platform supports comprehensive analysis of low-abundance PTMs.
2. Advanced Mass Spectrometry Capabilities
Platforms such as Orbitrap Exploris 480 and timsTOF Pro 2 support both DDA and DIA acquisition modes with exceptional sensitivity.
3. Custom Enrichment Strategies
Tailored solutions for lactylated peptide enrichment that balance specificity and detection sensitivity.
4. In-Depth Bioinformatics Support
Personalized analysis of signaling pathways, transcriptional regulatory networks, and delivery of fully visualized analytical reports.
As a critical intersection point between metabolism and epigenetic regulation, lactylation is gaining increasing scientific attention. LFQ provides a powerful, flexible, and sample-efficient approach for precise detection and quantification of lactylated proteins. At MtoZ Biolabs, we are committed to continuously refining our proteomics platforms to empower researchers in transitioning from PTM mapping to mechanistic understanding. For inquiries regarding lactylated proteomics or other PTM-focused research, please contact MtoZ Biolabs for expert consultation and customized solutions.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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