Principle of Protein Deamidation Analysis

Protein post-translational modifications (PTMs) are crucial mechanisms that regulate protein functionality and structural diversity. Among these, deamidation refers to the conversion of asparagine (Asn) or glutamine (Gln) residues into aspartic acid (Asp) or glutamic acid (Glu), respectively. This modification alters protein structure and stability, and it is closely associated with the development of various diseases, making its precise analysis critically important.

 

Deamidation typically occurs spontaneously under non-enzymatic conditions. The amide groups in asparagine or glutamine residues are hydrolyzed to form carboxyl groups, resulting in changes to the primary structure of the protein. This process depends on the surrounding amino acid environment and factors such as pH and temperature. Deamidation can lead to alterations in protein charge, conformation, and surface properties, thereby impacting protein function.

 

Technical Principles of Protein Deamidation Analysis

Mass spectrometry (MS) is the primary tool for analyzing protein deamidation. MS detects deamidation by measuring the mass difference in peptide fragments. For instance, converting an asparagine residue to an aspartic acid residue results in a 0.984 Da mass increase. The mass spectrometer can precisely measure this subtle mass difference, enabling the identification of deamidated proteins.

 

1. Sample Preparation

The protein sample is first digested into peptides, typically using trypsin. To enhance the detection sensitivity of deamidation, the sample often undergoes enrichment steps, such as separating deamidated peptides using liquid chromatography (LC).

 

2. Mass Spectrometry Analysis

The peptides are then ionized and introduced into the mass spectrometer via electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). By measuring the mass-to-charge ratio (m/z) of the peptides, the mass spectrometer detects the mass differences caused by deamidation. In the mass spectrum, the signal peak of the peptide will shift compared to the unmodified peptide, indicating deamidation.

 

3. Data Analysis

Mass spectrometry data is typically analyzed using database searches or de novo sequencing methods. Database searches match experimental data with theoretical spectra, identifying peptides potentially modified by deamidation. De novo sequencing infers peptide sequences and modification sites directly from mass spectrometry data without relying on databases.

 

Mass spectrometry has become the core tool for studying protein deamidation due to its efficiency and accuracy. However, the complexity of deamidation presents challenges, such as precise localization of modification sites and detection of low-abundance modifications. As mass spectrometry technology and data analysis methods continue to advance, protein deamidation analysis will more accurately reveal its role in biological processes.