Histone Ubiquitination Analysis

Histones are a highly conserved class of proteins, with only minor differences in amino acid sequences between their various variants. The N-terminal region of histones can undergo several types of modifications, including methylation, acetylation, phosphorylation, ubiquitination, and ADP-ribosylation. These covalent modifications are fundamentally involved in regulating gene transcription, DNA repair, replication, and chromosome condensation. Among these, histone ubiquitination represents a critical post-translational modification (PTM), characterized by the covalent attachment of ubiquitin molecules to specific lysine residues on histones.

 



Liu, R. et al. MedComm. 2023.

Figure 1. Biological Effects Mediated by Histone Modification

 

Histone ubiquitination is crucial for understanding epigenetic regulation. It serves as an essential mechanism for modulating gene expression and is also closely associated with the onset and progression of numerous diseases. In particular, abnormal ubiquitination is frequently linked to genomic instability and tumorigenesis in cancer research. Consequently, a deeper investigation into histone ubiquitination may elucidate its role in disease pathogenesis and offer potential therapeutic targets for conditions such as cancer.

 

Services at MtoZ Biolabs

MtoZ Biolabs utilizes Thermo Fisher's Q Exactive HF, Orbitrap Fusion, and Orbitrap Fusion Lumos mass spectrometry platforms, in combination with Nano-LC, to offer a comprehensive solution for histone ubiquitination analysis. Simply provide us with your experimental objectives and sample materials, and we will manage the entire workflow, delivering detailed identification and quantitative analysis of histone ubiquitination modifications and their modification sites. Compared to conventional quantitative proteomic analyses, the key to histone modification analysis lies in the isolation and purification of histone components and mitigating the impact of N-terminal lysine on histone post-translational modification analysis. MtoZ Biolabs proposes countermeasures based on literature and existing experience:

 

1. The Isolation and Purification of Histone

Since histones are located in the cell nucleus, MtoZ Biolabs has developed a specialized platform for histone extraction and purification, drawing on established literature and experimental expertise. After homogenizing the cells, nuclei are separated via low-speed centrifugation. The nuclei are then lysed to release histones, which are further isolated from DNA. Impurities are removed using high-salt buffer precipitation, and HPLC is employed to purify the histones of interest, which are subsequently subjected to mass spectrometry analysis.

 

2. Solutions to Mitigating the Impact of N-Terminal Lysine on Histone PTM

Furthermore, to eliminate the impact of N-terminal lysine, we employ 2-3 different enzymes during the proteolytic digestion process. This approach improves peptide coverage in mass spectrometry, reducing the loss of histone PTM information due to suboptimal peptide length or low ionization efficiency.

 

Analysis Workflow

 



 

Service Advantages

1. Advance Analysis Platform: Mtoz Biolabs established an advanced histone ubiquitination analysis platform, guaranteeing reliable, fast, and highly accurate analysis service.

 

2. One-Time-Charge: Our pricing is transparent, no hidden fees or additional costs.

 

3. High-Data-Quality: Deep data coverage with strict data quality control. AI-powered bioinformatics platform integrate all histone ubiquitination analysis data providing clients with acomprehensive data report.

 

4. Optimized Protocol: An optimized histone extraction and purification protocol is employed to ensure high-quality samples, preventing degradation and contamination, as well as optimized solutions to mitigating the impact of N-terminal lysine on histone PTM.

 

Sample Submission Requirements

1. If you are submitting tissue samples, please ensure that they are shipped on dry ice. For plant tissues, the sample weight should exceed 400 mg; for blood samples, a minimum volume of 2 mL is required (plasma samples should be treated with EDTA as an anticoagulant); for serum, at least 2 mL is necessary; for urine, a volume of 10 mL is required; animal tissue samples should weigh no less than 2 g, and cell samples should contain a minimum of 1 x 10⁸ cells. For yeast and microbial samples, please submit a minimum dry weight of 400 mg.

   

2. If you are providing protein samples, please ensure that the total protein amount falls within the range of 2-5 mg. Standard tissue or cell lysis buffers can be used during protein extraction.

   

3. For sample shipment, use an adequate amount of dry ice and choose expedited shipping methods to minimize the risk of sample degradation during transit.

 

4. Prior to initiating the formal analysis, we will conduct quality checks on the provided samples. Only after the samples pass our quality assessment will the experimental procedures commence.

 

Deliverables

1. Comprehensive Experimental Details

2. Materials, Instruments, and Methods

3. Relevant Liquid Chromatography and Mass Spectrometry Parameters

4. The Detailed Information of Histone Ubiquitination Analysis

5. Mass Spectrometry Image

6. Bioinformatics Analysis

7. Raw Data

 

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider, provides advanced proteomics, metabolomics, and biopharmaceutical analysis services to researchers in biochemistry, biotechnology, and biopharmaceutical fields. Our ultimate aim is to provide more rapid, high-throughput, and cost-effective analysis, with exceptional data quality and minimal sample consumption. If you are interested in our histone ubiquitination analysis service, please feel free to contact us. We are dedicated to providing you with the best service.