Dimethyl Labeling

Dimethyl labeling is an efficient chemical labeling method widely used in quantitative proteomics research. This method is based on the chemical modification of the N-terminus of peptides and the side chain amino groups of lysine residues. It utilizes formaldehyde and cyanoborohydride under alkaline conditions to perform reductive amination, achieving light, medium, and heavy isotopic labeling of peptides. The main advantages of this technique include its simplicity, high labeling efficiency, applicability to complex biological samples, and compatibility with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for high-throughput and precise protein quantification. In proteomics research, dimethyl labeling can be used to compare protein expression levels across different samples, such as in disease research, drug mechanism exploration, and cell signaling pathway analysis. Compared to other quantitative methods (such as SILAC, iTRAQ, TMT, etc.), this technology offers low cost, high throughput, and relatively simple experimental procedures, making it particularly suitable for large-scale protein quantification. In recent years, with the advancement of mass spectrometry technology, dimethyl labeling has become a tool for protein quantification research, providing an efficient and reliable quantitative strategy for biomedical research. Despite its excellent performance in quantitative proteomics research, dimethyl labeling still has some limitations. For instance, due to its reliance on lysine residues, proteins with low lysine content may be challenging to quantify accurately, affecting data comprehensiveness. Additionally, in experiments requiring extremely high mass accuracy, slight retention time shifts of different isotopically labeled dimethyl groups may affect quantitative precision.

 

1. Principle of Dimethyl Labeling

The basic principle of dimethyl labeling is based on the reductive amination reaction of amino groups. Under alkaline conditions, formaldehyde reacts with the N-terminus and the ε-amino group of lysine residues in peptides to form unstable imines (Schiff bases), which are then reduced by cyanoborohydride to form stable dimethyl modifications. By choosing formaldehyde labeled with different isotopes (¹²CH₂O, ¹³CD₂O, etc.), light, medium, and heavy labeled peptides can be obtained, allowing different samples to be distinguished in the same mass spectrometry analysis and quantified by mass spectrometry signal intensity ratios. Compared to other chemical labeling methods, dimethyl labeling offers highly controllable labeling sites and typically does not affect peptide ionization efficiency or mass spectrometric behavior. Additionally, this method is applicable to complex protein samples, including tissues, plasma, and cell lysates, making it highly suitable for proteomics research.

 

2. Experimental Workflow of Dimethyl Labeling

The experimental workflow of dimethyl labeling mainly includes five key steps: sample preparation, protein digestion, chemical labeling, sample mixing, and mass spectrometry analysis.

1. Sample Preparation: Extract proteins from cells, tissues, or body fluids, and remove insoluble impurities using methods such as sonication and centrifugation.

2. Protein Digestion: Use enzymes such as trypsin or Lys-C to hydrolyze proteins into peptides to expose specific labeling sites (N-terminus and lysine side chains) for dimethyl labeling.

3. Dimethyl Labeling: Add isotopically labeled formaldehyde and cyanoborohydride under alkaline conditions to chemically modify the peptides.

4. Sample Mixing: Mix different labeled samples in equal proportions to ensure the accuracy of mass spectrometry analysis.

5. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) Analysis: Use high-performance liquid chromatography to separate peptides and detect the relative abundance of different isotopically labeled peptides through mass spectrometry for quantitative analysis.

 

Biotech Pack offers high-quality SILAC/Dimethyl labeling-based quantitative proteomics analysis services, dedicated to providing researchers with efficient and precise protein quantification solutions. Our experimental team strictly controls labeling conditions to ensure high labeling efficiency and data reliability.

 

Biotech Pack - Characterization of Biological Products, Premier Multi-omics Mass Spectrometry Service Provider

 

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SILAC/Dimethyl Labeling-Based Quantitative Proteomics Analysis