Principle of Quantitative Acetylproteomics

Post-translational modifications (PTMs) play a crucial role in regulating cellular functions. Among them, acetylation is one of the most common, impacting protein structure, function, and interactions with other molecules. Quantitative acetyl proteomics involves the high-throughput analysis of acetylated proteins and their changes under various physiological conditions, shedding light on their biological functions. This technique, which combines mass spectrometry (MS) with protein quantification methods, provides key insights into the role of acetylation in biological processes like diseases and cell signaling.

 

The core principle of quantitative acetyl proteomics lies in detecting and quantifying the acetylation modifications of specific proteins using mass spectrometry. The basic steps include:

 

1. Protein Extraction and Acetylated Peptide Enrichment

Proteins in the sample are broken down into peptides through appropriate lysis and digestion steps. Specialized antibodies or affinity tags are then used to selectively enrich acetylated peptides, ensuring specificity and sensitivity in subsequent analyses.

 

2. Mass Spectrometry Analysis

The enriched acetylated peptides are separated and identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS provides information on peptide molecular weight and amino acid sequence, which helps pinpoint the exact location of acetylation sites. Quantification of the MS data is achieved through either labeling techniques (e.g., SILAC or TMT) or label-free methods (e.g., DIA).

 

3. Data Analysis and Quantification

The quantification of MS data can be performed in two ways: labeled quantification or label-free quantification. Labeled quantification involves comparing acetylation levels between different samples using isotopically labeled peptides, while label-free quantification is based on relative comparisons of MS signal intensities. Data analysis software (e.g., MaxQuant) searches and matches the MS data to a database, identifying acetylation sites and quantifying them.

 

Biological Significance of Acetylation

Acetylation plays significant roles in various biological processes, including gene expression regulation, cell cycle control, and metabolic regulation. For instance, histone acetylation can alter chromatin structure, thereby influencing gene transcription activity. Additionally, non-histone acetylation has been widely reported in proteins such as metabolic enzymes and transcription factors, affecting their catalytic activity, stability, and protein-protein interactions.

 

Applications of Quantitative Acetyl Proteomics

Quantitative acetyl proteomics is widely applied in disease research, drug development, and basic biological studies. In particular, in cancer and neurodegenerative diseases, dynamic changes in acetylation modifications can help identify novel therapeutic targets and biomarkers.

 

By combining high-throughput mass spectrometry techniques with precise quantification methods, researchers can comprehensively analyze the role of protein acetylation in biological functions. The application of this technique is driving our understanding of cellular regulatory mechanisms and offering new insights for clinical research.