SILAC/Dimethyl

SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) is a mass spectrometry-based quantitative proteomics technique. By incorporating stable isotope-labeled amino acids into cells during culture, SILAC enables precise quantification of proteins in mass spectrometry analysis. The core principle involves adding labeled amino acids, such as ¹³C₆-lysine or ¹³C₆,¹⁵N₄-arginine, to the culture medium, allowing cells to integrate these labeled amino acids into newly synthesized proteins. During mass spectrometry, the difference in mass between labeled and unlabeled proteins, due to the isotope incorporation, facilitates the comparison of protein expression levels under different conditions. SILAC is particularly suitable for quantifying proteins in cell culture systems, including mammalian, yeast, and certain prokaryotic cells. Generally, full-length proteins synthesized within cells are ideal for this quantification method.

 

Dimethyl quantitative proteomics is another mass spectrometry-based quantitative proteomics technique. This method achieves relative quantification of proteins by chemically labeling proteins or peptides using dimethyl labels. The core principle involves labeling the N-terminus and lysine side chains of proteins or peptides with dimethyl labeling reagents (typically formaldehyde and cyanoborohydride). By incorporating different isotope labels (such as light, medium, and heavy labels) during sample preparation, the relative abundance of proteins in different samples can be distinguished and compared in mass spectrometry analysis. Dimethyl quantitative proteomics uses different isotope forms of formaldehyde (such as ¹²C-formaldehyde, ¹³C-formaldehyde) and cyanoborohydride for dimethyl labeling, followed by detection of labeled peptides using liquid chromatography-mass spectrometry (LC-MS/MS). This technique is particularly suitable for relative quantification of proteins or peptides in complex samples, such as cell lysates and tissue samples, and can be applied to almost all proteins and peptides due to the efficient labeling of the N-terminus and lysine residues.

 

Dimethyl Quantitative Proteomics Analysis Workflow

1. Sample Preparation: Lyse and digest protein samples from different treatment conditions into peptides.

2. Dimethyl Labeling: Chemically label peptides with isotope-labeled formaldehyde and cyanoborohydride.

3. Sample Mixing: Combine the differently labeled samples to ensure simultaneous detection in mass spectrometry analysis.

4. Mass Spectrometry Analysis: Separate and detect the mixed samples using LC-MS/MS.

5. Data Analysis: Process mass spectrometry data with specialized software to compare the relative abundance of different labeled peptides.

 

SILAC Quantitative Proteomics Analysis Workflow

1. Cell Culture: Culture control and experimental group cells in media containing normal and labeled amino acids, respectively.

2. Cell Lysis and Protein Extraction: Collect, lyse the cells, and extract total proteins.

3. Protein Digestion: Digest the proteins into peptides using trypsin or other proteases.

4. Mass Spectrometry Analysis: Separate and detect the peptides using LC-MS/MS.

5. Data Analysis: Analyze mass spectrometry data using specialized software to compare the relative abundance of labeled and unlabeled peptides.



Chen, X. et al. Proteomics. 2015.

Figure 1. Workflow for Quantitative Proteomic Experiments Using SILAC

 



Hsu, JL. et al. Philos Trans A Math Phys Eng Sci. 2016.

Figure 2. Labeling Scheme for Stable Isotope Dimethyl Labeling

 

Applications

1. Protein Expression Analysis: Compare protein expression changes under different conditions to reveal the functional and regulatory roles of proteins in biological processes.

2. Protein-Protein Interaction Studies: Identify compositional changes in protein complexes under specific physiological or pathological conditions.

3. Post-Translational Modification Studies: Quantify changes in specific modified proteins, such as phosphorylation and acetylation.

4. Proteome-Wide Protein Turnover Studies: Investigate the dynamics of protein turnover at the proteome level.

5. Biomarker Discovery: Identify potential biomarkers in disease or specific physiological conditions.

 

Service Advantages

1. High Precision: The stability of isotope labeling and the high resolution of mass spectrometry ensure highly accurate quantification.

2. Low Variation: Culturing the same cell line under identical conditions minimizes biological and technical variability.

3. Simplicity: SILAC does not require chemical labeling or additional processing, reducing experimental complexity and potential errors.

4. High Throughput: Capable of labeling and analyzing multiple samples simultaneously, suitable for large-scale proteomics research.

5. High Sensitivity: Dimethyl labeling has high labeling efficiency and sensitivity, enabling the detection of low-abundance proteins.

6. Low Cost: Dimethyl labeling reagents are relatively inexpensive and easy to use, suitable for routine laboratory experiments.

7. Versatility: Applicable to almost all proteins and peptides, with a wide range of applications.

 

Due to their high accuracy and broad applicability, SILAC and Dimethyl Quantitative Proteomics have become important tools in quantitative proteomics research. MtoZ Biolabs is dedicated to advancing proteomics research, continuously updating our technologies and instruments to provide researchers with efficient and precise proteomics services. For inquiries about our services, please contact us.