Histone PTMs Quantitative Analysis Service

Histones, as core structural components of chromatin, play essential roles in regulating gene expression, DNA repair, chromatin remodeling, and epigenetic control. The N-terminal tails and core regions of histones are subject to a variety of post-translational modifications (PTMs), including methylation, acetylation, phosphorylation, ubiquitination, and SUMOylation. These PTMs modulate chromatin structure or recruit specific reader proteins, thereby orchestrating diverse nuclear functions with high precision.

 



Huang, H. et al. Chem Rev. 2015.

Figure 1. Structures of Histone Post-Translational Modifications

 

Researchers are increasingly recognizing the regulatory importance of histone PTMs in cell fate determination and disease progression, particularly in cancer, stem cell differentiation, immune responses, and neurological disorders. Traditional techniques such as Western blotting and ChIP can detect certain modifications, but are limited in throughput, sensitivity, and quantification accuracy. The advent of high-resolution mass spectrometry (MS) enables comprehensive and quantitative analysis of histone modifications.

 

MtoZ Biolabs leverages advanced mass spectrometry platforms and optimized protocols for histone extraction, derivatization, and enrichment to offer Histone PTMs Quantitative Analysis Service. This service provides researchers with detailed and reliable chromatin modification profiles, facilitating the advancement of epigenetic research.

 

Technical Principles

Histone PTMs quantitative analysis refers to the precise detection of specific histone modification types, abundance levels, and dynamic changes across different biological conditions using high-resolution mass spectrometry. This analysis typically adopts one or more of the following strategies:

 

1. Bottom-up MS: Digests histones into short peptides before MS/MS analysis; suitable for high-throughput detection of multiple PTM sites but may lose combinatorial context.

2. Middle-down MS: Retains longer peptides for a balance between site identification and PTM pattern recognition.

3. Top-down MS: Analyzes intact histones directly, allowing for detection of complex combinatorial PTMs, though it requires higher-end instruments and is suited to specific research goals.

 

Depending on the study design, quantification can be achieved using stable isotope labeling methods (e.g., SILAC, TMT) or label-free approaches. Nano-flow liquid chromatography (nanoLC) is employed for high-resolution peptide separation, ensuring accurate detection of low-abundance modifications.

 



Karch, K.R. et al. Methods Enzymol. 2016. 

Figure 2. Workflows for Bottom-Up, Middle-Down, and Top-Down Histone PTM Analysis by High-Resolution Tandem MS

 

Analysis Workflow

MtoZ Biolabs has developed a standardized yet flexible analytical workflow that covers all steps from sample processing to data delivery:

1. Sample Preparation

Histones are extracted from cells or tissues using high-salt or acid extraction methods to ensure sample purity and PTM preservation.

 

2. Protein Pre-Treatment

Includes reduction, alkylation, and chemical derivatization (e.g., propionylation) to block non-specific sites and improve site identification.

 

3. Enzymatic Digestion

Proteolysis using enzymes such as trypsin, Arg-C, or Glu-C depending on the PTM type to produce MS-compatible peptides.

 

4. NanoLC Separation

Peptides are separated with high resolution via nanoLC to enhance detection sensitivity for modified peptides.

 

5. Mass Spectrometry (MS/MS)

Peptides are analyzed using high-resolution mass spectrometers (e.g., Orbitrap, Q-TOF) to identify peptide sequences and modification sites.

 

6. Data Processing and Quantification

Specialized software is used for PTM site identification, intensity extraction, quantitative calculations, and differential analysis.

 

7. Report and Data Output

A full report is delivered, including quantitative tables, PTM maps, functional annotations, and data visualizations.

 

Service Advantages

1. Multi-platform Support: Compatible with multiple analytical strategies tailored to various PTM types and research needs.

2. Comprehensive PTM Coverage: Covers common and novel modifications on histones H3, H4, H2A, and H2B.

3. High Sensitivity: Optimized enrichment and separation workflows enhance detection of low-abundance PTMs.

4. Flexible Quantification Options: Supports SILAC, TMT, and label-free quantification.

5. Standardized Quality Control: Full traceability and data reliability ensured from sample to analysis.

6. Sample Versatility: Applicable to a wide range of sample types, including cell lines, tissue specimens, blood, and FFPE blocks.

 

Applications

The Histone PTMs Quantitative Analysis Service can be applied to a variety of research areas:

1. Epigenetics: Elucidating the link between chromatin modifications and gene regulation.

2. Cancer Biology: Profiling abnormal histone modification patterns in cancer to identify potential therapeutic targets.

3. Cell Cycle Regulation: Monitoring PTM dynamics across different cell cycle phases.

4. Development and Differentiation: Revealing PTM signatures in specific tissues or developmental stages.

5. Drug Screening: Evaluating the impact of epigenetic-targeted compounds on histone modifications.

6. Neuroscience: Investigating histone PTM alterations in neurons or brain tissue.

 

Case Study

1. A Quantitative Atlas of Histone Modification Signatures from Human Cancer Cells

This study systematically analyzed single and combinatorial post-translational modifications (PTMs) of histones H3 and H4 across 24 human cancer cell lines. By integrating transcriptomic data to assess the expression of histone-modifying enzymes, the researchers revealed that certain PTMs were regulated independently of enzyme expression. Notably, H3K27 methylation was highly enriched in breast cancer cell lines. EZH2 knockdown experiments in a mouse mammary xenograft model confirmed the critical role of histone modifications in tumor progression and highlighted the utility of proteomics in epigenetic cancer classification. Histone PTMs Quantitative Analysis Service enables precise quantification of multi-site and combinatorial histone modifications, helping researchers explore chromatin states and epigenetic regulation across different cell types. The service covers a wide range of cancer cell models and is suitable for studies on tumor classification, enzyme activity inference, and functional validation.

 



Leroy, G. et al. Epigenetics Chromatin. 2013.

Figure 3. Proteomic Strategy for Histone PTM Quantification

 

2. Quantitative Analysis of Post-Translational Modifications of Histone H3 Variants During the Cell Cycle

This study established a quantitative MRM-SIS mass spectrometry method using stable isotope-labeled internal standards to precisely analyze the post-translational modification (PTM) patterns and abundance changes of three major histone H3 variants in Karpas 422 cells across different cell cycle stages. The results revealed distinct dynamic methylation changes at K27 and K36 among different H3 variants, with H3.3 showing an opposite K36me2 trend compared to H3.1 and H3.2. These findings highlight the unique regulatory roles of histone variants in epigenetic control during the cell cycle. Histone PTMs Quantitative Analysis Service enables dynamic profiling of histone variant modifications across cell cycle phases, supporting investigations into site-specific regulation and functional differences among histone subtypes. The service accommodates high-throughput workflows to advance in-depth mechanistic studies.

 



Chen, J. et al. Anal Chim Acta. 2019.

Figure 4. Quantitative Analysis of Modifications on Lysine 27 and 36 in H3.1, H3.2 and H3.3. Modification on K27-R40 During the Cell Cycle in H3.1, H3.2, H3.3

 

FAQ

Q1: What sample types are supported?

A1: Supported sample types include cells, animal tissues, frozen or FFPE tissues, and clinical specimens. Please contact our technical team for guidance.

 

Q2: Can low-abundance modifications be detected?

A2: Yes. Our workflow incorporates enrichment and enhancement strategies to ensure reliable detection of low-abundance PTMs. A recommended minimum input amount should be ensured.

 

Q3: Can multiple modification types be analyzed simultaneously?

A3: Yes. The service supports simultaneous detection of various PTMs and analysis of combinatorial modification patterns.

 

Q4: What is the turnaround time?

A4: The typical turnaround is 2–4 weeks, depending on the sample number and analysis depth. Expedited service is available upon request.

 

Q5: Does the service include bioinformatics analysis?

A5: Yes. Basic analysis includes PTM site annotation and expression visualization. Extended services such as pathway enrichment and network analysis are also available.

 

If you are interested in learning more about the Histone PTMs Quantitative Analysis Service, including sample submission requirements or deliverable formats, please contact MtoZ Biolabs' technical support team. We are committed to providing high-quality, reliable analytical support to advance your epigenetic research.