Histone Acetylation Modification Mass Spectrometry Analysis

Histone acetylation is a crucial post-translational modification (PTM) that plays a key role in the regulation of gene expression, chromatin remodeling, and other cellular functions. Mass spectrometry has emerged as an essential tool for the identification and quantification of histone acetylation modifications. The following briefly describes the basic process of histone acetylation mass spectrometry analysis:

 

Analysis Workflow

1. Sample Preparation

(1) Extract histones from cells or tissues.

(2) Use enzymes such as trypsin or lipase to digest histones, obtaining histone peptides.

 

2. Enrichment of Acetylated Peptides

(1) Use antibodies with affinity or other methods to enrich peptides with acetylation modifications.

 

3. Liquid Chromatography Tandem Mass Spectrometry Analysis (LC-MS/MS)

(1) Use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze the enriched samples.

(2) The mass spectrometer can accurately identify and quantify peptides with acetylation modifications.

 

4. Data Processing and Analysis

(1) Use specialized mass spectrometry data processing software, such as MaxQuant, Skyline, or other tools, to process data, identify acetylation modification sites, and perform quantitative analysis.

(2) Perform bioinformatic analysis, including function, pathway, and network analysis, to reveal the role of acetylation in biological processes.

 

5. Experimental Verification

(1) For key or research focus acetylation sites, other methods (such as Western blotting, immunofluorescence staining, etc.) can be used for verification.

 

6. Precautions

(1) During the sample processing process, it is necessary to avoid protein degradation or the occurrence of non-specific modifications as much as possible.

(2) Since acetylation modification is reversible, the sample processing should be done as quickly as possible and avoid long-term exposure under non-physiological conditions.