Workflow of SILAC and Dimethyl Labeling in Quantitative Proteomics

Quantitative proteomics often relies on methods such as SILAC (Stable Isotope Labeling by Amino acids in Cell culture) and dimethyl labeling for comparing the relative abundance of proteins across different samples. Each method has distinct advantages, making them suitable for various experimental conditions.

 

SILAC Workflow

1. Sample Preparation

In the SILAC approach, cells are cultured in media containing either light or heavy isotope-labeled amino acids, such as lysine (Lys) and arginine (Arg). These labeled amino acids are incorporated into newly synthesized proteins, effectively labeling the proteins within the cells.

 

2. Protein Extraction and Digestion

Following the cell culture, the cells are lysed to extract the total protein content. The extracted proteins are then enzymatically digested into peptides, commonly using trypsin. These peptides will include both light and heavy forms, corresponding to the light and heavy isotopes used.

 

3. Sample Mixing and Mass Spectrometry Analysis

The resulting peptides, labeled with light and heavy isotopes, are mixed in equal proportions and subjected to mass spectrometry analysis. The mass spectrometer distinguishes between the isotope-labeled peptides, enabling the quantification of protein abundance across the different samples.

 

4. Data Analysis

Mass spectrometry data provides the basis for calculating the relative abundance of proteins between the sample groups. These quantitative results are crucial for understanding protein functions, interactions, and differential expression under various conditions.

 

Dimethyl Labeling Workflow

1. Sample Labeling

Dimethyl labeling, a versatile chemical labeling technique, is applicable to a broad range of sample types. The workflow begins with the extraction and enzymatic digestion of protein samples into peptides. These peptides are then labeled at the N-terminus and lysine residues using dimethyl labeling reagents, creating light, medium, and heavy isotope-labeled forms.

 

2. Sample Mixing and Mass Spectrometry Analysis

The differently labeled peptides are combined in equal proportions (1:1:1) and analyzed using mass spectrometry. The mass spectrometer detects and distinguishes between the light, medium, and heavy-labeled peptides, facilitating the quantification of protein abundance across different samples.

 

3. Data Analysis

The mass spectrometry data reflect the abundance ratios of the differently labeled peptides. By analyzing these ratios, researchers can accurately assess the relative changes in protein expression under different experimental conditions.

 

Both SILAC and dimethyl labeling offer unique benefits and limitations. SILAC is particularly well-suited for live cell studies, allowing for isotopic labeling without altering protein properties. In contrast, dimethyl labeling is more adaptable, applicable to a broader array of sample types.