Targeted Protein Degradation (TPD) analysis with FIDA

Guide to Assaying Ternary Complex Formation for Targeted Protein Degradation Using Fida Instrument

‍

Watch the video to learn how to use the Fida instrument for assaying ternary complex formation in targeted protein degradation.

Terminology:

• Indicator: Fluorescent molecules whose size is measured.
• Analyte: The compound that is titrated in the assay.

Fida Experiment Methods:

1. Premix Method:
2. Involves pre-mixing equal amounts of indicator and analyte in vials before injection.
3. Capillary Mix (Cap Mix) Method:
4. Employs a single indicator vial with a titration of analyte vials. The mixing happens in the capillary by the Fida instrument.
5. Capillary Dissociation (Cap Dis) Method:
6. Involves a titration of mixed indicator and analyte vials with a single buffer vial.
7. Capillary Flow (CapFLEX) Method:
8. Optional method, mainly for liquid phase separation and condensate analysis.

Targeted Protein Degradation:

• Bispecific Molecules:
• Utilised to facilitate ternary complex formation between a target protein and ubiquitin E3 ligase, leading to proteosomal degradation of the target.
• Governed by two equilibrium constants and their cooperativity.
• Full characterisation of biophysical parameters possible with Fida.
• Molecular Glues:
• Modifies the target protein surface to enhance interaction with the E3 ligase.
• Characterised by a single affinity constant, simplifying the assay process.

Experimental Setup:

• In Cell Lysate Assays:
• Recommended to use the premix method for assays directly in cell lysate.
• Equal amounts of analyte and indicator are mixed.
• Characterisation Using Bispecific Molecules:
• Perform either a cap mix or a premix experiment.
• Essential to ensure correct concentration range to avoid overshooting ternary complex formation due to the hook effect.

Data Analysis Using Fida Software:

1. Loading and Evaluating Data:
2. Load data files and select the appropriate time interval for analysis.
3. Data Fitting:
4. Choose single or multispecies fitting based on the experiment.
5. Auto mode available for efficient batch fitting of data points.
6. Manually evaluate and correct each fit as needed.
7. Finalising Analysis:
8. Compensate for viscosity and check fluorescence areas.
9. Select the ternary complex model in the software and input initial guesses for parameters.
10. Apply and fit the binding curve to view the results.

Conclusion:This guide provides a thorough walkthrough for conducting and analysing ternary complex assays for targeted protein degradation using the Fida instrument. It is designed to enhance your understanding and assist in achieving precise and reliable results in your research.

Final Note:We hope this guide proves beneficial in your scientific endeavours. For visual assistance, please refer to our accompanying video.