Acetylation Detection Methods

Protein acetylation is a critical protein post-translational modification, which involves the covalent binding of an acetyl group to a lysine residue by an acetyl donor (such as acetyl coenzyme A) through enzymatic or non-enzymatic means. Currently, two forms of acetylation are known - Nα acetylation and Nε acetylation. Nα acetylation refers to the acetylation modification of the N-terminus of a protein, which is generally considered irreversible, very common in eukaryotes, present in nearly 85% of eukaryotic proteins, and very rare in prokaryotes. In contrast to Nα acetylation, Nε acetylation is dynamic and reversible, mainly occurring in histones, high-mobility group (HMG) proteins, transcription factors, nuclear receptors, and α-tubulin.

 

Research on protein acetylation has a history of over 60 years and has now become a hotspot in the international protein field.

 

1. Early Technology

Early acetylated protein detection methods mainly include immunoaffinity detection and radioactive detection. They use specific antibodies to identify acetylated proteins, with radioactive detection being more suitable for in vivo labelled or in vitro enzymatically synthesized acetylated proteins. However, these methods cannot provide specific information about acetylation modification sites.

 

2. Commonly Used Technology Today

Combination of two-dimensional electrophoresis and immunoblotting: Using two-dimensional electrophoresis to separate target proteins, then probe treatment with antibodies that can specifically identify acetylated proteins. These antibodies will bind to acetylated proteins to form antigen-antibody complexes. Then, by adding labelled secondary antibodies (such as horseradish peroxidase or fluorescence dye labelled antibodies) to detect these complexes, the presence and distribution of acetylated proteins can be visualized.

 

Characteristics: High specificity sensitivity; however, it is technically demanding and complex in operation.

 

3. Mass Spectrometry Technology

First, label cellular proteins with isotopes of different molecular weights during cell culture (SILAC), then enrich acetylated peptides by immuno-precipitation after protein digestion, and finally combine chromatographic separation techniques and mass spectrometry techniques (HPLC/MS) to realize qualitative and quantitative research on acetylation modification.

 

Characteristics: High sensitivity resolution, can be used in combination with various techniques; however, it is time-consuming, complex to operate, and difficult to apply on a large scale.

 

4. Protein Chip Technology

Fix capture antibodies on a substrate material, capture target proteins through the interaction between target proteins and capture antibodies, and then use pre-labelled detection antibodies or markers to identify target molecules and perform quantitative analysis.

 

Characteristics: High throughput, high specificity and sensitivity, and very small sample consumption; however, the cost is relatively high, the technical system is incomplete, and there is a lack of uniformity.