Dimethyl Labeling
Dimethyl labeling is a highly efficient chemical labeling technique widely used in quantitative proteomics. This method relies on the chemical modification of amine groups on peptide N-termini and lysine side chains through reductive amination using formaldehyde and sodium cyanoborohydride under alkaline conditions. This reaction generates peptides with three distinct mass labels—light, medium, and heavy—allowing simultaneous differentiation and quantification of samples within a single mass spectrometry analysis.
A key advantage of dimethyl labeling is its simplicity, high labeling efficiency, and compatibility with complex biological samples. When coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it enables high-throughput and accurate protein quantification. This method is extensively applied in comparative proteomic studies, including disease biomarker identification, drug mechanism investigations, and cellular signaling pathway analyses. Compared with other quantitative proteomic techniques such as SILAC, iTRAQ, and TMT, dimethyl labeling offers lower cost, higher throughput, and a streamlined workflow, making it particularly advantageous for large-scale protein quantification studies.
Despite its advantages, dimethyl labeling has certain limitations. Since labeling depends on lysine residues, proteins with low lysine content may be difficult to quantify accurately, potentially leading to incomplete quantitative data. Furthermore, in experiments requiring ultra-high mass accuracy, minor retention time shifts may occur between different isotopically labeled dimethyl groups, potentially affecting quantification precision.
Principle of Dimethyl Labeling
Dimethyl labeling is based on the reductive amination of amines. Under alkaline conditions, formaldehyde reacts with peptide N-termini and lysine ε-amino groups to form unstable imines (Schiff bases), which are subsequently reduced to stable dimethyl-modified peptides by sodium cyanoborohydride. Using isotopically labeled formaldehyde (¹²CH₂O, ¹³CD₂O, etc.), peptides with distinct light, medium, and heavy mass tags can be generated, enabling differentiation of samples in a single mass spectrometry run and allowing protein quantification based on signal intensity ratios.
Experimental Workflow
1. Sample Preparation
Proteins are extracted from cells, tissues, or biological fluids, followed by removal of insoluble impurities through sonication and centrifugation.
2. Protein Digestion
Proteins are enzymatically digested with trypsin or Lys-C to expose labeling sites.
3. Dimethyl Labeling
Peptides are chemically labeled under alkaline conditions using isotopically labeled formaldehyde and sodium cyanoborohydride.
4. Sample Mixing
Labeled samples are mixed in equal proportions to ensure quantitative accuracy.
5. LC-MS/MS Analysis
Peptides are separated via liquid chromatography and analyzed by mass spectrometry to determine relative abundances.
MtoZ Biolabs offers high-quality SILAC/Dimethyl labeling-based quantitative proteomics services, ensuring high labeling efficiency and reliable data for researchers.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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