Step-by-Step Protocol: How to Perform a DARTS Assay

Drug Affinity Responsive Target Stability (DARTS) is a powerful, label-free chemical proteomics technique designed to identify and validate the protein targets of small molecules based on their ability to confer proteolytic resistance. Unlike traditional affinity-based pulldown methods, DARTS exploits the conformational changes that occur upon ligand binding—changes that increase a protein's resistance to proteolysis.

DARTS has gained traction in early-phase drug discovery, particularly for compounds lacking reactive handles or for complex biological matrices where direct affinity tagging is impractical. This protocol provides a comprehensive, scientifically rigorous, and execution-ready guide for researchers aiming to integrate DARTS into their experimental toolkit.

Principle of the DARTS Assay

The DARTS assay leverages a biophysical phenomenon wherein ligand binding induces conformational stabilization of a target protein. When a small molecule binds to its protein partner, it often results in reduced structural flexibility, shielding specific protease cleavage sites and thereby conferring resistance to proteolytic digestion. By comparing the proteolytic profiles of ligand-treated and vehicle-treated samples, researchers can infer target engagement.

Central to the assay's success is the careful modulation of proteolysis. Over-digestion may obscure differential stabilization, while under-digestion may not reveal meaningful differences between treated and untreated samples. The choice of protease is also critical; enzymes such as pronase—a mixture of broad-specificity proteases—are often preferred for their ability to degrade a wide range of proteins under mild conditions. Alternatively, more selective proteases like thermolysin or subtilisin may be employed when a narrower cleavage profile is desired.

Interpretation of DARTS results typically involves either:

• Qualitative analysis, where the appearance or disappearance of protein bands on SDS-PAGE indicates ligand protection;
• Or Quantitative analysis, where densitometric measurement of band intensity differences is used to calculate stabilization levels.

When integrated with orthogonal approaches such as mass spectrometry (DARTS-MS), DARTS can evolve from a targeted validation tool into a robust, discovery-driven platform, enabling unbiased identification of novel drug targets.

Materials and Reagents

A meticulous selection of materials and reagents is essential to ensure the reliability and reproducibility of the DARTS assay. Below is a breakdown of the necessary components, categorized for clarity:

A. Biological Materials

• Cultured Cell Lines: Commonly used lines such as HEK293, HeLa, or NIH/3T3 cells are ideal due to their robust protein expression profiles and ease of handling. Cells should be harvested during the log phase of growth to maintain protein integrity and ensure reproducibility.
• Tissue Samples: For in vivo relevance, fresh or flash-frozen tissues (e.g., liver, brain, or tumor biopsies) can be used. Tissue homogenization should be performed under cold conditions to prevent proteolysis prior to assay setup.
• Purified Proteins: Recombinant or native purified proteins allow for mechanistic studies under controlled conditions, minimizing background interference from complex lysates.

B. Buffers and Reagents

Lysis Buffer Composition:

• 50 mM HEPES, pH 7.4: Maintains physiological pH critical for preserving protein conformation.
• 150 mM NaCl: Ensures isotonic conditions to minimize osmotic shock during lysis.
• 1% Triton X-100 or NP-40: Non-ionic detergents used to solubilize membrane proteins while maintaining protein-protein interactions.
• Protease Inhibitor Cocktail (EDTA-free): Optional; added only after digestion step to prevent non-specific degradation during storage.

Note: Detergents such as SDS must be avoided in lysis buffers as they denature proteins, abolishing ligand-induced stabilization.

Proteases:

• Pronase: A cocktail of proteolytic enzymes with broad substrate specificity, suitable for initial optimization.
• Thermolysin or Subtilisin: For selective digestion based on specific amino acid motifs or secondary structure.

Quantitation and Analysis Kits:

• BCA Protein Assay Kit: Offers accurate and detergent-compatible quantification of total protein concentration.
• SDS-PAGE and Western Blotting Reagents: Includes pre-cast gels, running buffer, transfer buffer, and blocking agents.

C. Instrumentation

Microcentrifuge: Capable of achieving ≥14,000 × g for efficient lysate clarification.

Electrophoresis System: Reliable power supplies and gel apparatus for consistent protein separation.

Western Blotting Setup: Semi-dry or wet transfer systems for efficient protein immobilization onto membranes.

Detection Systems:

• Chemiluminescence Imagers (e.g., Bio-Rad ChemiDoc)
• Fluorescence Imagers (e.g., LI-COR Odyssey) for multiplexed or quantitative analyses.

Ensuring the use of high-quality, consistent reagents and calibrated instrumentation will significantly enhance assay sensitivity, minimize variability, and facilitate the generation of publication-grade data.

Experimental Design Considerations

Designing a DARTS experiment requires careful optimization of multiple parameters to ensure specificity, sensitivity, and reproducibility. Unlike routine biochemical assays, DARTS experiments are acutely sensitive to variations in protease concentration, protein stability, and ligand binding conditions.

Biological Replicates

To achieve statistical significance, a minimum of three independent biological replicates is mandatory. These replicates must originate from separately prepared lysates to account for biological variability. For particularly heterogeneous samples, such as primary tissues, increasing the number of replicates is advisable to enhance robustness.

Compound Preparation

The physicochemical properties of the small molecule under investigation—such as solubility, stability, and aggregation propensity—must be considered:

• Solvent Choice: DMSO is the solvent of choice for most small molecules; however, the final DMSO concentration in the assay must not exceed 1% (v/v) to prevent nonspecific effects on protein structure.
• Compound Stock: Prepare highly concentrated stock solutions (e.g., 10–100 mM) to minimize dilution effects and solvent impact.
• Handling: Minimize freeze-thaw cycles of compounds to avoid precipitation or degradation.

Control Design

Appropriate controls are critical for meaningful interpretation:

• Vehicle Control: Essential to distinguish compound-specific effects from vehicle-related artifacts.
• Positive Control: If available, use a molecule known to bind the target to validate assay responsiveness.
• No-Protease Control: Provides baseline information on total protein input and verifies sample loading consistency across lanes.

Protein Concentration Optimization

Lysates must be standardized to a consistent protein concentration, typically 1–3 mg/mL. Suboptimal protein concentrations can either dilute potential target signals or saturate the protease, masking differential stabilization.

Additional Parameters

• Temperature: Incubation temperature should align with the biological relevance of the interaction; generally, room temperature (20–25°C) or physiological temperature (37°C) is appropriate.
• Time Points: Consider multiple time points for protease digestion to build a kinetic profile of stabilization.
• Protease Titration: Pre-experiment optimization is crucial to find the ‘sweet spot' where partial digestion reveals stabilization without complete degradation.

By meticulously planning these variables, the probability of generating clean, interpretable, and reproducible DARTS results dramatically increases, ultimately reducing experimental iterations and accelerating discovery timelines.

Step-by-Step Experimental Protocol for DARTS Assay

Executing a DARTS assay requires careful attention at each stage to preserve the native conformations of proteins and to maintain the integrity of ligand-protein interactions. The following stepwise guide is structured to facilitate precise and reproducible implementation:

1. Lysate Preparation

Procedure:

1) Harvesting:

For cultured cells: Grow cells to ~80% confluency. Rinse with cold PBS, scrape, and pellet by centrifugation at 500 × g for 5 minutes at 4°C.

For tissue samples: Dissect fresh or thaw flash-frozen tissue quickly on ice. Minimize time outside cold conditions to prevent spontaneous proteolysis.

2) Cell/Tissue Lysis:

Resuspend the cell pellet or minced tissue in 5 volumes of ice-cold lysis buffer.

Homogenize:

• For cells: Pipette up and down or pass through a 27G needle multiple times.
• For tissues: Use a dounce homogenizer or mechanical disruptor under cold conditions.

3) Clarification:

Centrifuge lysates at 14,000 × g for 15 minutes at 4°C.

Transfer the clear supernatant to a fresh pre-chilled tube without disturbing the pellet.

4) Protein Quantification:

Determine protein concentration using the BCA assay.

Normalize all samples to an identical final concentration (typically 2 mg/mL).

Aliquot lysates appropriately and keep on ice for immediate use, or flash-freeze for short-term storage at −80°C.

Critical Tips:

• Avoid using detergents like SDS during lysis, as they denature proteins and abolish native binding sites.
• Maintain lysates on ice at all times to preserve native conformations and prevent premature proteolysis.

2. Compound Incubation

Procedure:

1) Sample Division:

Split normalized lysates into two groups: Compound-treated and Vehicle-treated.

Ensure equal volumes and protein concentrations across all samples to guarantee comparability.

2) Compound Addition:

Add the small molecule to the treatment group to reach the desired final concentration (commonly 1–10 μM, depending on the expected binding affinity).

For the vehicle control, add an equivalent volume of solvent (e.g., DMSO) to match the compound-treated group's final solvent concentration (typically ≤1%).

3) Incubation Conditions:

Gently mix the samples to ensure uniform distribution of the compound.

Incubate:

• At room temperature (20–25°C) for standard protein-ligand interactions, or
• At 4°C if the ligand or proteins are unstable at higher temperatures.

4) Optional:

For weak or slow-binding compounds, prolong incubation times (up to 2–4 hours) or perform a short thermal stabilization step (e.g., incubation at 37°C for 15 minutes) prior to proteolysis.

Critical Tips:

• Ensure complete solubilization of the compound before addition to lysates; incomplete dissolution can lead to false negatives.
• Minimize freeze-thaw cycles of compounds to preserve potency and binding capacity.
• Use low-binding tubes to avoid adsorption of hydrophobic compounds to tube walls.

3. Protease Digestion

Procedure:

1) Preparation of Protease Stock:

Dilute the selected protease (e.g., pronase) freshly in reaction buffer (e.g., PBS or HEPES buffer) immediately before use.

Typical working dilutions range from 1:300 to 1:500 (enzyme:lysate, v/v), but empirical optimization is essential to fine-tune digestion intensity.

2) Initiating Digestion:

Add an appropriate amount of protease to each sample (compound-treated and vehicle-treated).

Mix gently but thoroughly to ensure homogenous distribution of the protease throughout the lysate.

3) Incubation:

Incubate the samples at 37°C, maintaining gentle agitation if possible (e.g., slow orbital shaking).

Set a time course if desired, commonly including 0, 10, 20, and 30-minute time points, to monitor progressive digestion.

For each time point, prepare a separate aliquot or stop the reaction sequentially.

4) Stopping the Reaction:

Immediately quench digestion by adding 4× SDS-PAGE sample buffer (Laemmli buffer) directly to the reaction mixture.

Boil the samples at 95°C for 5 minutes to fully denature the proteins and halt proteolytic activity.

Critical Tips:

• Optimization is mandatory: Perform a preliminary protease titration with vehicle-treated lysates alone to determine the digestion conditions where partial degradation is visible but not complete destruction.
• Protease activity is temperature-sensitive: Always pre-warm reaction components to avoid delays that could introduce variability.
• Timing is crucial: Especially for short digestion periods (<15 minutes), stagger sample setup carefully to ensure exact incubation times.

4. SDS-PAGE and Western Blot

Procedure:

1) Preparation of SDS-PAGE Gel:

Prepare or purchase a 10–12% SDS-PAGE gel, depending on the molecular weight range of the target proteins.

Use a 4–20% gradient gel if the molecular weight distribution is broad.

Assemble the gel and prepare the running buffer (e.g., 1× Tris-Glycine SDS buffer) according to standard protocols.

2) Loading Samples:

Mix the quenching reaction (lysate + protease) with 4× SDS-PAGE sample buffer (containing β-mercaptoethanol for reducing conditions) to ensure denaturation of the proteins.

Load equal volumes of each sample (e.g., 30–40 µL per lane) into the gel wells. Ensure that the total protein amount across samples is consistent to allow for meaningful comparisons.

3) Electrophoresis:

Run the gel at constant voltage (typically 120–150 V) until the dye front reaches the bottom of the gel (approximately 1–1.5 hours, depending on gel thickness and buffer system).

To ensure adequate resolution, adjust the voltage if necessary, especially for larger proteins or if using gradient gels.

4) Transfer to Membrane:

After electrophoresis, transfer the proteins from the gel to a PVDF membrane using a wet transfer (or semi-dry transfer system) at 100 V for 1 hour or follow specific transfer conditions based on membrane type and protein size.

Pre-wet the PVDF membrane in 100% methanol for 1–2 minutes before transfer to activate it, followed by washing in transfer buffer.

5) Blocking:

Block the membrane with 5% non-fat dry milk in 1× TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature or overnight at 4°C to reduce non-specific binding.

6) Incubation with Primary Antibody:

Incubate the membrane with a primary antibody specific for your target protein (e.g., GAPDH for loading control or specific binding partners) in TBST containing 5% BSA or milk.

Incubate overnight at 4°C with gentle agitation or for 1–2 hours at room temperature.

7) Secondary Antibody Incubation:

After washing the membrane with TBST, incubate with the appropriate HRP-conjugated secondary antibody for 1 hour at room temperature.

Dilution ratios for secondary antibodies should be optimized (usually 1:10,000 to 1:50,000).

8) Detection:

Wash the membrane thoroughly to remove excess secondary antibody (typically 3 × 5 minutes in TBST).

Develop the blot using an ECL chemiluminescent substrate or fluorescent detection system.

Capture the signal using a chemiluminescence imager or fluorescent scanner.

Critical Tips:

• Ensure membrane activation: Proper pre-wetting of PVDF membranes with methanol is essential for effective protein binding.
• Blocking buffer choice: Non-fat dry milk is typically used, but in some cases (such as when dealing with high background), BSA (bovine serum albumin) may be preferable.
• Signal clarity: Optimal exposure times are critical to avoid saturation. If using chemiluminescence, start with a shorter exposure time and increase if necessary.

a Schematic of target identification of ATP, PEP and L-Phe with DARTS. b Workflow of target fishing and MS identification. c Workflow of chemical proteomics combined with protein microarray (Chen, Xiao, et al., 2020)

Data Interpretation for DARTS Assay

Qualitative Analysis (Visual Inspection):

After completing the Western blot, assess the protein bands corresponding to the target protein(s) in both the compound-treated and vehicle-treated samples.

Look for differences in band intensity:

• Protection from digestion: Bands in the compound-treated samples that appear stronger (or remain intact) compared to the vehicle-treated control suggest that the small molecule has stabilized the target protein.
• Degradation pattern: A faster degradation or weaker band in vehicle-treated samples indicates that the protease is effectively cleaving the protein in the absence of ligand binding.

Quantitative Analysis (Densitometry):

For more precise and quantitative data, use densitometry software (e.g., ImageJ, Li-Cor Odyssey, or commercial chemiluminescence software) to analyze the band intensities. Calculate the relative intensities of the target bands from both the compound-treated and vehicle-treated samples at various protease digestion time points.

The relative protection index (RPI) can be derived by comparing the band intensities at different protease treatment times:

A higher RPI indicates stronger stabilization and more significant target engagement by the compound.

Statistical Analysis:

Conduct statistical analysis on the data to assess the significance of the observed differences in protein stability between the treated and control groups.

Use statistical tests like Student's t-test for pairwise comparisons (vehicle vs. compound), or ANOVA if comparing multiple conditions (e.g., different doses or different time points).

Comparison to Controls:

• No-protease control: A sample where protease treatment is omitted should show the total amount of protein (unaffected by proteolysis), serving as a baseline for sample loading efficiency and protein stability.
• Positive control: If available, use a compound with a known mechanism of action (e.g., a ligand known to bind the target protein) to validate the assay's capacity to detect target stabilization.

Advanced Data Analysis:

When using mass spectrometry (DARTS-MS), compare the relative abundance of identified peptides in compound-treated versus vehicle-treated samples. An increase in peptide intensity in the compound-treated samples relative to vehicle samples indicates ligand-induced stabilization.

Utilize bioinformatics tools to map out peptide regions protected by the ligand, which can provide insight into potential binding sites or conformational changes induced by compound binding.

Critical Tips:

• Control validation: Ensure that vehicle-treated samples show substantial degradation under protease treatment, which acts as a baseline to confirm the assay's effectiveness.
• Avoid overexposure: For chemiluminescence detection, overexposure of the blot can result in saturation, leading to inaccurate intensity measurements.
• Multiple replicates: Always perform multiple biological replicates (at least three) to ensure the robustness of your data and mitigate the effects of random error.

Troubleshooting and Best Practices

Despite the power of the DARTS assay, challenges may arise during experimentation. Here, we address common issues and provide solutions to optimize experimental outcomes.

Weak or No Signal in Western Blot

Possible Causes:

• Low protein input: If the initial protein concentration in the lysate is too low, the signal may be weak or absent.
• Improper blocking conditions: Inadequate blocking (e.g., using the wrong blocking buffer or insufficient blocking time) can lead to high background or weak signals.
• Antibody issues: Low-affinity or improperly diluted primary antibodies can fail to detect the target protein, or secondary antibodies may not be optimal for the chosen detection system.

Solutions:

• Increase protein loading: Ensure adequate protein concentration, typically 2–3 mg/mL, for the lysate before loading onto the gel. If necessary, increase the volume of sample loaded.
• Optimize antibody dilution: Test a range of dilutions for both primary and secondary antibodies. Refer to the manufacturer's recommended conditions and adjust according to your protein of interest.
• Enhance blocking efficiency: Consider using higher concentrations of blocking agents (e.g., 5% BSA instead of milk for high-affinity targets) or extend blocking time to ensure complete membrane coverage.
• Check primary antibody quality: Validate primary antibody specificity and ensure it recognizes the protein in the lysate by running a positive control.

No Differential Protection Between Compound-treated and Vehicle-treated Samples

Possible Causes:

• Inadequate compound concentration: If the small molecule does not bind effectively to the target protein, it will not induce stabilization, leading to no observable protection.
• Insufficient incubation time: The ligand-protein interaction may require longer incubation periods to reach equilibrium.
• Protease over-digestion: If the protease is too active or the digestion time is too long, all proteins may be degraded regardless of ligand binding, obscuring the stabilization effects.

Solutions:

• Increase compound concentration: Perform dose-response experiments to identify the optimal concentration for ligand binding. Often, concentrations in the low micromolar range (1–10 µM) are effective.
• Prolong incubation: For weak-binding ligands, increase the incubation period (up to 2–4 hours) to allow for more effective binding and stabilization.
• Optimize protease digestion: Reduce protease incubation times and perform a titration to determine the optimal protease concentration for partial digestion.

High Background in Western Blot

Possible Causes:

• Non-specific binding: High background can occur if the primary antibody or secondary antibody binds non-specifically to proteins or other components on the membrane.
• Improper washing: Insufficient washing between antibody incubations can lead to residual antibodies, increasing background.
• Protein contamination: Contaminants in lysates or from the protease treatment may contribute to non-specific signals.

Solutions:

• Enhance washing steps: Increase the stringency of washing with TBST (increased number of washes or longer wash times) to remove non-specific antibody binding.
• Reduce antibody concentrations: Use lower antibody concentrations during incubation and check if this reduces background without compromising signal strength.
• Use pre-adsorbed antibodies: Some antibodies are available pre-adsorbed to reduce non-specific binding. Alternatively, pre-adsorb antibodies with lysate from a non-relevant source (e.g., tissue or cell type) before use.
• Use blocking alternatives: Try using other blocking reagents such as Superblock™ or BSA if blocking with milk leads to high background.

Inconsistent Protease Activity Across Replicates

Possible Causes:

• Protease lot-to-lot variability: Different batches of proteases may have different levels of activity, leading to inconsistent results.
• Storage issues: Improper storage of proteases (e.g., repeated freeze-thaw cycles) can reduce their activity.

Solutions:

• Test protease activity: Perform a titration of the protease on lysates without any compound treatment to determine optimal digestion conditions.
• Use fresh protease: Prepare fresh protease stock solutions for each experiment to minimize degradation over time.
• Check storage conditions: Ensure proteases are stored according to manufacturer guidelines, typically in aliquots at −20°C to avoid multiple freeze-thaw cycles.

Problems with Mass Spectrometry Integration (DARTS-MS)

Possible Causes:

• Low peptide recovery: Loss of peptides during proteolysis, sample preparation, or analysis may hinder detection.
• Matrix interference: Matrix components or high-abundance proteins may interfere with peptide detection, leading to noisy spectra or missing data.
• Inadequate peptide labeling or digestion: If peptides are not sufficiently digested or if digestion times are inconsistent, the data quality will suffer.

Solutions:

• Optimize digestion time and conditions: Experiment with different protease concentrations, incubation times, and buffer compositions to ensure complete but not over-digested samples.
• Use tryptic digestion: In cases of poor proteolysis with broad-specificity proteases, consider using a more specific enzyme such as trypsin to generate more consistent and predictable peptide patterns.
• Clean-up samples: Use solid-phase extraction (SPE) or size-exclusion chromatography to remove interfering matrix components before mass spectrometric analysis.
• Standardize instrumentation settings: Ensure that mass spectrometer calibration and parameters (e.g., collision energy, scan rate) are optimized for your specific peptides of interest.

References

1. Lomenick, Brett, et al. "Target identification using drug affinity responsive target stability (DARTS)." Proceedings of the National Academy of Sciences 106.51 (2009): 21984-21989. https://doi.org/10.1073/pnas.0910040106
2. Martinez Molina, D. & Nordlund, P. (2016). The cellular thermal shift assay: A novel biophysical assay for in situ drug target engagement and mechanistic biomarker studies. Annu Rev Pharmacol Toxicol 56, 141–161. https://doi.org/10.1146/annurev-pharmtox-010715-103715
3. Chen, Xiao, et al. "Target identification of natural medicine with chemical proteomics approach: probe synthesis, target fishing and protein identification." Signal Transduction and Targeted Therapy 5.1 (2020): 72. https://doi.org/10.1016/j.bcp.2021.114798