The effect of Oxidation on TTR Amyloid aggregation

I was extremely lucky to have been accepted into the National Amyloidosis Centre to do a lab research project on whether oxidation increases the rate of TTR amyloid aggregation in vitro. As part of my project, I was specifically comparing the V122I mutant (known to be very aggregation prone) with wild-type TTR. The proteins were made using recombinant bacteria which was already prepared by my supervisor before I started the project. My role was to prepare a physiological environment for both proteins using phosphate buffer saline (PBS) which is slightly alkaline. After I prepared both samples I used a spectrophotometer to measure the concentration of proteins in solution using the Beer-Lambert Equation.

At this point, I was able to calculate the amount of copper chloride (control) and hydrogen peroxide (experimental) needed for protein oxidation. We used a copper chloride, THT, trypsin solution to create an oxidising environment for the degraded proteins and measured light absorption to see how many fibrils aggregated. From our investigation, we have noticed increased aggregation in the oxidising environment compared to the control. We noted that trypsin could have been replaced with plasmin in a future experiment to reflect on the more ubiquitous enzyme in vivo.

 

In order to confirm no false-positives, I used congo red staining to observe amyloid under the microscope after preparing the slides.
Before aggregation, we tested the purity of the samples using LC-MS by comparing the M/Z ratios to the reference values for the corresponding wild-type and mutant TTR. The samples were first separated into fragment peptides using liquid chromatography with a gradient of buffer A (hydrophilic) and buffer B (hydrophobic), followed by mass spectrometry analysis to measure the mass-to-charge ratios.
In the video, 2 µL of the protein sample is taken for analysis, and 18 µL of solvent (e.g., a buffer or reagent) is added to dilute the sample. This step ensures that the protein concentration is within the optimal range for the machine. After we collected the results, we noticed that some of our initial proteins may have already underwent some oxidation. There was a smaller peak after the m/z of our TTR protein of 14034, suggestive of oxygen being added:
After aggregation, another mass spec confirmed with the MASCOT database (with the local database from the centre) that there was increased oxidation particularly on residues (methionine, cysteine, histidine, tryptophan) of beta-pleated sheets.

I also assessed the impact of oxidation on protein stability by treating both WT and V122I variants with increasing concentrations of denaturants. No significant difference in tetramer stability was observed, as indicated by similar levels of monomer release.

Conclusion:

This project suggests that the enhanced aggregation seen with the oxidised V122I variant is not due to increased dissociation but may instead reflect the involvement of additional aggregation-prone intermediates—such as dimers or partially unfolded species—that were not resolved by our 4–20% SDS-PAGE. These findings imply that oxidation influences the aggregation pathway without significantly altering the stability of the native tetramer.