Mass Spectrometry to find a therapeutic drug for liver disease and COPD

Summary

Do Chelani discusses their work in Dr. Constantinos Thalasinos' lab, where they focus on identifying the binding affinities of a Nova tetrapeptide to Alpha 1-antitrypsin and its mutants. Using mass spectrometry, they aim to determine whether the tetrapeptide could be a therapeutic agent for diseases like COPD caused by Alpha 1-antitrypsin aggregation. Chelani describes the process of preparing samples, creating gold-coated electro spray needles, and analyzing data. The hands-on experience with mass spectrometry has been the most enjoyable aspect of their project, providing valuable insights into protein interactions and therapeutic potential.

Takeaways

• 😀 Do Chelani works in Dr. Constantinos Thalasinos' lab, focusing on mass spectrometry in structural biology.
• 😀 The project aims to identify the binding affinities of a Nova tetrapeptide to alpha-1 antitrypsin and its mutant, exploring its potential as a therapeutic agent.
• 😀 The goal is to prevent aggregation of alpha-1 antitrypsin, which is linked to diseases like emphysema and COPD.
• 😀 Mass spectrometry is used to compare protein samples in bound and unbound states to determine binding conditions.
• 😀 Needle pulling from silicate glass capillaries is an essential part of the preparation process for mass spectrometry.
• 😀 The capillary tubes are gold-coated to prevent oxidation and ensure proper electrospray ionization during the analysis.
• 😀 A vacuum chamber is used to replace oxygen with argon to avoid reactions that could interfere with gold coating.
• 😀 After coating, the needles are ready to be used for electrospray ionization in mass spectrometry experiments.
• 😀 Protein samples are purified using membrane and gel filtration techniques and then loaded into a volatile buffer (ammonium acetate).
• 😀 Technical replicates are needed to obtain reliable data, which is collected into mass spectra and analyzed for accuracy.
• 😀 Do Chelani enjoys working directly with mass spectrometry, as it enhances understanding and provides hands-on experience with the technology.

Q & A

What is the primary objective of Do Chelani's project?

The primary objective is to identify the binding affinities of a Nova tetrapeptide to Alpha 1 antitrypsin and its mutants, to determine whether it could be a potential therapeutic agent.

How does the therapeutic agent in Do's project relate to diseases like COPD?

The therapeutic agent aims to stop the aggregation of Alpha 1 antitrypsin, which causes diseases like emphysema and pulmonary disorders such as COPD.

What is mass spectrometry, and how is it used in this project?

Mass spectrometry is a technique that compares the mass-to-charge ratios of bound and unbound protein samples to determine binding conditions. In this project, it's used to analyze the interaction between the tetrapeptide and Alpha 1 antitrypsin.

What is the role of the needle puller in the process?

The needle puller is used to create electrospray needles by pulling capillary tubes from silicate glass, which are then gold-coated and used for sample analysis in the mass spectrometer.

Why is it necessary to replace oxygen with argon during the gold coating process?

Argon is used in the gold coating process to prevent oxygen from reacting with the gold when the capillary tubes are coated, ensuring a smooth and stable coating.

What is the purpose of the mass spectrometer in the project?

The mass spectrometer is used to analyze the protein samples and collect data on their mass spectra, which helps determine the binding affinity between the tetrapeptide and Alpha 1 antitrypsin.

What steps are involved in preparing the protein sample for analysis?

The protein sample must first be purified and placed in a volatile buffer, such as ammonium acetate. Techniques like membrane and gel filtration are used to purify the sample before it's loaded into the mass spectrometer.

Why are technical replicates required in the project?

Technical replicates are required to ensure reliable and consistent data, as they help account for any variation in the measurements and improve the accuracy of the results.

What aspect of the project does Do Chelani find most enjoyable?

Do Chelani finds working with the mass spectrometer the most enjoyable, especially because it allows for hands-on experience and a deeper understanding of how the instrument produces data.

What is the significance of the gold coating on the capillary tubes?

The gold coating on the capillary tubes is essential for the electrospray process, as it helps in the efficient transfer of the protein sample into the mass spectrometer, ensuring accurate data collection.