What Are the DARTS Steps? Is Mass Spectrometry After Staining or Target Identification? How if Stained Proteins Change?
The DARTS technique involves several key steps, as outlined below:
Sample Preparation
The drug to be tested is mixed with proteins from cell lysates or tissue extracts, and incubated for a specified period.
Protein Digestion
After incubation, a protease (typically trypsin) is added for partial digestion of the proteins.
Gel Electrophoresis
The digested samples are analyzed using SDS-PAGE to separate protein fragments based on size.
Mass Spectrometry Analysis
After electrophoresis, the protein bands are excised, typically selecting those that show significant differences when compared to the control group, and then subjected to mass spectrometry to identify the proteins in these bands.
In the DARTS workflow, mass spectrometry can be performed either before or after staining, depending on the experimental objectives:
Direct Mass Spectrometry from Unstained Gel Slices
This method is the standard procedure, used to identify potential drug target proteins directly from the DARTS experiment. In this case, proteins are separated via SDS-PAGE, and bands containing potential target proteins are excised directly from the unstained gel for mass spectrometry analysis. The advantage of this approach is that it eliminates the potential chemical contamination introduced by staining, thus ensuring the accuracy and sensitivity of mass spectrometry analysis.
During this step, mass spectrometry is used to identify proteins that exhibit altered stability in the presence or absence of the drug. These proteins show enhanced stability in the presence of the drug, as evidenced by their reduced digestion during proteolysis.
Post-Staining Mass Spectrometry for Further Protein Verification
In some cases, Coomassie Brilliant Blue staining is first performed to visualize the overall distribution of proteins in the sample. Specific bands of interest are then excised for further mass spectrometry analysis. This is typically done after preliminary data suggests potential drug target proteins. Staining helps to confirm and optimize the experiment, particularly in refining protein extraction and digestion efficiency.
While post-staining mass spectrometry may present some challenges, such as potential interference from dye residues, modern protein purification and elution techniques are sufficient to overcome these issues, ensuring that proteins from stained gels remain suitable for high-quality mass spectrometry analysis.
In cases where multiple proteins show changes in staining patterns, it suggests that several proteins may be responsive to the drug. In such instances, proteins exhibiting pronounced stability changes in the drug-treated samples (e.g., more prominent bands or proteins less digested) should be selected for mass spectrometry analysis. This allows for more targeted identification of potential drug-interacting target proteins.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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