Specific Chemical Labeling of Protein Lysine Methacrylation: Mechanisms, Methods and Research Tools

Concept

Protein lysine methacrylation (Kmcra) is a newly discovered post-translational acylation modification and a structural isomer of lysine crotonylation (Kcr). Distinguished by the unique position of its carbon-carbon double bond in the acyl side chain, methacrylation is derived from specific acyl-CoA metabolites and is widely distributed in core histones and non-histone proteins. It participates in the regulation of key biological processes including chromatin remodeling, gene expression, mitochondrial metabolism, and cell cycle control, and is closely associated with the pathogenesis of certain genetic and metabolic diseases. Specific chemical labeling of methacrylation refers to the selective covalent modification and tagging of methacrylated lysine residues in proteins using chemical strategies, enabling the enrichment, detection and identification of this modification in complex biological systems—an essential technical foundation for exploring its biological functions and regulatory mechanisms.

Research Frontiers

The research of protein lysine methacrylation is a rapidly evolving frontier in the field of post-translational modification (PTM) biology, with key research frontiers focused on solving technical bottlenecks and exploring biological functions:

1. Development of specific labeling and enrichment technologies: Addressing the technical challenge of distinguishing methacrylation from its structural isomer crotonylation, the latest research focuses on chemical strategies based on structural differences (spatial hindrance, radical intermediate stability) to achieve selective labeling, replacing traditional antibody-based methods with severe cross-reactivity issues.
2. Photocatalytic chemical reaction design for biological systems: The application of photocatalytic thiol-Michael addition reactions in PTM specific labeling has become a research hotspot, with optimized design of water-soluble, cell-compatible small molecule probes to ensure efficient and specific reactions in complex physiological environments.
3. High-throughput proteomic identification of methacrylation substrates: Combining specific chemical labeling with click chemistry and high-resolution mass spectrometry to establish a complete proteomic workflow, enabling systematic identification of novel methacrylated proteins and precise modification sites in histones and non-histones.
4. Exploration of methacrylation in metabolic-epigenetic crosstalk: A core research frontier is clarifying how methacrylation, as a metabolite-derived modification, mediates the regulation of epigenetic processes (e.g., histone function, gene expression) by metabolic signals, and its dynamic changes under different physiological and pathological conditions.
5. Development of high-specificity research tools: The engineering of recombinant monoclonal antibodies targeting methacrylation with minimal cross-reactivity to structural analog modifications (crotonylation, propionylation, butyrylation) has become a key direction for supporting in-depth research of this modification.

Research Significance

Methacrylation, as a novel metabolite-linked acylation modification, has important research significance spanning chemical biology, epigenetics, metabolism and disease research, and the development of specific chemical labeling technologies for this modification further unlocks its scientific value:

1. Filling the technical gap in specific detection of methacrylation: The structural similarity between methacrylation and crotonylation has long hindered accurate identification of the former; specific chemical labeling technologies break this bottleneck, providing a reliable technical means for the qualitative and quantitative analysis of methacrylation in biological samples.
2. Expanding the understanding of acylation modification networks: Specific labeling and proteomic identification enable the discovery of a large number of novel methacrylated substrates and modification sites, expanding the scope of known methacrylation modification networks and revealing its unique regulatory roles distinct from other acylation modifications.
3. Uncovering the molecular mechanism of metabolic-epigenetic crosstalk: As a modification derived from acyl-CoA metabolites, methacrylation is a key molecular link between cellular metabolism and epigenetic regulation; its specific detection helps clarify how metabolic changes regulate epigenetic processes and cell fate decisions, enriching the theory of metabolic-epigenetic interaction.
4. Providing new research clues for metabolic disease mechanisms: Abnormal methacrylation modifications are associated with hereditary metabolic disorders and cancer metabolic reprogramming; specific labeling technologies enable the identification of disease-related abnormal methacrylation substrates and sites, providing potential molecular targets for the diagnosis and treatment of metabolic diseases.
5. Promoting the innovation of PTM research methods: The design strategy of specific chemical labeling for methacrylation (based on structural differences and photocatalytic reactions) provides a new paradigm for the research of other PTMs with structural analogs, driving the innovation of chemical biology methods in PTM research.

Mechanisms & Research Methods

Core Challenge: Specific Labeling of Methacrylation Against Structural Isomer Interference

Protein lysine methacrylation and crotonylation are structural isomers, differing only in the position of the carbon-carbon double bond in the acyl group, and both are derived from acyl-CoA metabolites with overlapping cellular distribution. This high structural similarity leads to severe cross-reactivity of traditional pan-methacrylation antibodies with crotonylation signals, making it impossible to accurately distinguish and enrich methacrylated proteins/peptides. In complex biological samples, the high background of crotonylation further interferes with the identification of methacrylation modification sites, becoming the biggest technical bottleneck restricting the in-depth research of methacrylation’s biological functions. Therefore, developing chemical labeling strategies that can exploit subtle structural differences between the two modifications to achieve selective recognition is the core prerequisite for breaking through this research dilemma.

Molecular Design Strategy for Specific Chemical Labeling

The specific chemical labeling of methacrylation is based on a photocatalytic thiol-Michael addition reaction strategy, designed by exploiting the subtle differences in spatial hindrance and radical intermediate stability between methacrylamide and crotonamide structures:

1. Theoretical basis of structural selectivity: Theoretical calculations confirm that the carbon-carbon double bond of methacrylamide has lower thermodynamic energy barriers for thiol radical addition reactions and forms more stable secondary radical intermediates; in contrast, crotonamide has significant spatial hindrance effects and forms less stable radical intermediates, leading to extremely low reaction efficiency under the same conditions.
2. Design of small molecule probe: A water-soluble, cell-compatible small molecule probe is synthesized with two functional moieties:
• A benzenethiol reactive group at one end, which can be activated to generate thiol radicals under specific photocatalytic conditions, serving as the core group for selective addition to methacrylamide double bonds.
• A bio-orthogonal handle (azide group) at the other end, which provides a site for subsequent click chemistry reactions, enabling the connection of enrichment/detection tags (e.g., biotin) to the labeled modification sites.
3. Optimization of probe properties: The probe is designed with hydrophilic groups to ensure good water solubility, and its molecular size and charge are optimized to improve cellular compatibility, enabling its application in both in vitro protein samples and complex cell lysates.

Mechanism of Photocatalytic Thiol-Michael Addition for Specific Labeling

The specificity of methacrylation labeling is achieved through the precise regulation of photocatalytic thiol-Michael addition reactions, with the following core reaction mechanisms:

1. Photocatalytic activation of thiol groups: In the presence of a specific photocatalyst (e.g., organic photocatalysts with good biocompatibility), the benzenethiol group of the probe is activated under light irradiation to generate highly reactive thiol radicals, which are the key reactive species for the addition reaction.
2. Selective addition to methacrylation double bonds: The thiol radicals selectively attack the carbon-carbon double bond of methacrylated lysine residues on target proteins; the methyl group on the methacrylamide double bond provides appropriate spatial accessibility, and the reaction generates a stable secondary radical intermediate, which rapidly undergoes subsequent proton transfer to complete the covalent and irreversible linkage of the probe to the methacrylation site.
3. Reaction inefficiency for crotonylation: For crotonamide structures, the spatial hindrance of the acyl side chain prevents the thiol radical from effectively approaching the double bond, and the generated primary radical intermediate is unstable and easily quenched, resulting in almost no addition reaction under the same photocatalytic conditions.
4. Validation of reaction selectivity: In vitro experiments with model compounds and complex protein mixtures confirm that this labeling system achieves over 85% selectivity for methacrylation, and can efficiently label methacrylated residues even in the presence of high crotonylation background, with no significant cross-reactivity with other acylation modifications.

Proteomic Identification Workflow Based on Specific Chemical Labeling

A complete high-throughput proteomic identification workflow is established by combining specific chemical labeling with click chemistry, affinity enrichment and high-resolution mass spectrometry, enabling the systematic identification of methacrylated proteins and precise modification sites:

1. Probe labeling of biological samples: The small molecule probe is incubated with cell lysates or purified protein samples under photocatalytic conditions to achieve specific covalent labeling of methacrylated lysine residues.
2. Click chemistry biotinylation: The azide handle on the probe reacts with alkyne-modified biotin molecules through copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry, linking biotin tags to all methacrylation-labeled sites.
3. Affinity enrichment of methacrylated proteins/peptides: Streptavidin-modified magnetic beads are used to specifically capture biotinylated methacrylated proteins from complex whole-proteome backgrounds, achieving efficient enrichment of trace methacrylated components with high purity.
4. Enzymatic digestion and mass spectrometry analysis: The enriched methacrylated proteins are digested into peptides with trypsin, and the peptide mixture is analyzed by high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS).
5. Identification of modification sites and substrates: Bioinformatic analysis of mass spectrometry data is performed to identify methacrylated proteins and their precise lysine modification sites, and functional annotation of the identified substrates is carried out to explore the biological processes involved in methacrylation regulation.

This workflow overcomes the limitations of traditional antibody enrichment methods, and for the first time enables the systematic identification of novel methacrylation sites in both histones and non-histone proteins, greatly expanding the known methacrylation modification network.

Novel Biological Discoveries Enabled by Specific Labeling Technology

The application of specific chemical labeling technology has led to a series of important novel discoveries in methacrylation research, breaking through the technical limitations of traditional methods:

1. Precise mapping of histone methacrylation sites: The technology not only validates the known methacrylation modification regions in core histones (e.g., H3, H4), but also accurately locates multiple specific methacrylation sites on histone H3 that were not previously identified, clarifying the precise distribution of methacrylation on histones.
2. Discovery of novel non-histone methacrylation substrates: A large number of previously unknown non-histone methacrylated proteins are identified, which are involved in key biological processes including chromatin regulation, transcriptional activation, mitochondrial metabolism and cell cycle control, revealing the broad regulatory scope of methacrylation beyond epigenetic regulation.
3. Cross-validation of antibody and chemical labeling methods: Partial methacrylation sites identified by chemical labeling overlap with the results of traditional antibody methods, validating the reliability of the new technology; the majority of newly discovered sites complement the antibody-based results, significantly expanding the understanding of methacrylation modification profiles.
4. Clarification of methacrylation’s role in metabolic-epigenetic crosstalk: The identified methacrylated substrates include multiple metabolic enzymes and epigenetic regulatory proteins, providing direct molecular evidence for methacrylation as a key link mediating metabolic signal regulation of epigenetic processes, and laying a foundation for in-depth research on its regulatory mechanisms under physiological and pathological conditions.

Product Empowerment: ANT BIO’s High-Specificity Methacrylation Research Tool

To support the in-depth research of protein lysine methacrylation, ANT BIO PTE. LTD. has independently developed a high-performance recombinant mouse monoclonal antibody targeting methacrylation modifications—Methacryllysine Recombinant Mouse mAb (S-3455) (Catalog No.: S0B6453). Developed with advanced antibody engineering technology, this product achieves accurate and specific recognition of protein lysine methacrylation (Kmcra) modifications, and overcomes the cross-reactivity issues of traditional antibodies, providing a cutting-edge, reliable and easy-to-use research tool for the frontier field of methacrylation research. As a core product of the Starter sub-brand (ANT BIO’s antibody specialist), it is optimized for various immunoassay applications and fully meets the research needs of methacrylation substrate discovery, functional verification and omics analysis.

Core Advantages of Methacryllysine Recombinant Mouse mAb (S0B6453)

Key Application Scenarios for S0B6453

This high-specificity methacrylation antibody is suitable for a wide range of research applications in PTM biology, metabolism and epigenetics, and is the preferred tool for both basic research and translational research:

Professional Technical Support for S0B6453

ANT BIO provides a full set of professional technical support to ensure the efficient application of this methacrylation antibody in your research:

• Comprehensive product technical documentation: Including detailed immunogen sequence information, antibody characterization data, and strict specificity validation reports (cross-reactivity testing with other acylation modifications).
• Optimized experimental protocols: Recommended standard and optimized protocols for various applications (WB, IP, ChIP, IF/IHC, mIF/mIHC), including sample preparation, antibody dilution ratio, and detection conditions.
• Customized technical consultation: Our team of PTM research experts provides one-on-one technical consultation and experimental troubleshooting, supporting the solution of complex experimental problems in methacrylation research.
• Sample testing service: Free sample testing is available for pre-experimental verification, ensuring the antibody is suitable for your specific research system and experimental design.

PTM Research Empowerment Program: Exclusive Benefits for Methacrylation and PTM Research

To support researchers in overcoming experimental challenges in the frontier field of PTM research (including methacrylation), ANT BIO launches the PTM Research Empowerment Program with limited-time exclusive benefits, covering flagship PTM research products and professional academic resources:

Program Time

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Promotion Code

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Core Purchase Benefits

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Brand Mission

At ANT BIO PTE. LTD., our core mission is to empower life science research and biopharmaceutical development by providing high-quality, innovative and reliable biological reagents, research tools and comprehensive solutions. As a leading global provider of life science reagents, we have built three specialized sub-brands to cover the full spectrum of research needs, creating a one-stop procurement experience for researchers worldwide:

• Absin: Specializes in general life science reagents and kits, including cell culture consumables, biochemical reagents, immunoassay kits and basic experimental tools—your reliable partner for basic laboratory research.
• Starter: Our flagship antibody sub-brand, focusing on the R&D and production of high-specificity, high-affinity antibodies for PTM research, cancer research, neuroscience and immunology—including the methacrylation recombinant mAb S0B6453, the core tool for methacrylation research.
• UA: Dedicated to recombinant proteins and custom protein services, including recombinant cytokines, enzymes, antigen proteins and custom protein expression/purification services—supporting protein science, structural biology and drug discovery research.

We are committed to investing in R&D of cutting-edge research tools for frontier life science fields (such as novel PTM research), and providing professional technical support and academic resources to help researchers overcome experimental challenges and accelerate scientific breakthroughs. For ANT BIO, innovation is the core driving force, quality is the unshakable foundation, and customer-centricity is the eternal service concept.

Related Product List: Methacrylation and PTM Research Core Products

For detailed product specifications, pricing and stock information, please visit the ANT BIO official website or contact our sales team.

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ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.