Exploration of Methods for Solubility Studies of Amyloid Beta

Abstract

The protein amyloid beta (Aβ) is a major component in the amyloid plaques associated with Alzheimer’s disease. This project concerns the most common version of this protein, Aβ40. In a supersaturated solution, Aβ40 monomers can aggregate to form amyloid fibrils. The solubility of an amyloid protein is equal to the concentration of the monomeric peptide in solution at equilibrium. To study the solubility, with the ambition to better understand the driving forces of the fibril formation, there is a need for reliable methods to determine the monomer concentration at equilibrium. In this project, three such methods were explored.

The first quantification method utilised the fluorescent probe o-phtalaldehyde (OPA), which reacts with primary amines in the peptide. The second method was high-performance liquid chromatography with mass spectrometry (HPLC-MS), which gave results based on both UV absorbance and total ion count after separation based on hydrophobicity. Both OPA and HPLC-MS quantification required the monomers to be separated from the fibrils, prior to measurements. Additionally, the samples were incubated in plate readers with ThT prior to the separation and quantification. The third and final quantification method was nuclear magnetic resonance spectroscopy (NMR). In NMR, the concentration of the monomers was calculated both based on the initial concentration and on an external standard.

NMR reported the highest apparent solubility, OPA an intermediate value (with an increase as the total Aβ40 concentration increased) and HPLC-MS reported the lowest apparent solubility. Kinetic traps, adsorption and protein degradation/modification were suggested to contribute to the differences in the results. Future experiments should include incubating with shaking, measuring with NMR and then calculating with external standard. Furthermore, it is necessary to repeat the experiments and evaluate their accuracy and precision.

Popular Abstract

The increasing life expectancy in many countries is a wonderful thing, but it does also increase the urgency for us to prevent and treat disorders that occur in the late stages of life. Alzheimer’s disease, the most common form of dementia, is such a disorder. A defining feature of Alzheimer’s disease is the presence of plaques in the brain and these plaques are largely made up of clumps of a particular substance: the protein known as amyloid beta.

At high concentrations of amyloid beta, the protein molecules can fold and organise together into fiber-like structures, called amyloid fibrils. It is these fibrils that then can be deposited as plaques. My thesis project is concerned with the solubility of amyloid beta, which can here be understood as a measurement of how much the protein can accept to stay in solution, as opposed to forming the fibrils. Determining the solubility of amyloid beta at different conditions, such as different temperatures, can help us understand the driving forces in the formation of amyloid fibrils. This can in turn be helpful in trying to understand, and eventually prevent or treat, Alzheimer’s disease.

I explored three different ways to determine the solubility of amyloid beta. The first method used the chemical o-phtalaldehyde (OPA). When OPA is added to the protein, the solution gives of fluorescent light proportional to the amount of protein in the solution. The second method was high-performance liquid chromatography with mass spectrometry (HPLC-MS), where the amount of amyloid beta was determined based both on the absorbance of UV light and on the number of molecules with the mass corresponding to amyloid beta. The third and final method was nuclear magnetic resonance (NMR) spectroscopy. This method is based on how atoms behave differently in a magnetic field, depending on what atom it is and its chemical surroundings. By finding which signals are coming from atoms in the protein, the intensity of those signals was used to determine the concentration.

Out of the three methods that I explored, NMR reported the highest solubility, followed by OPA and with HPLC-MS giving the lowest value. While I cannot say which of these values is the closest to the real solubility, I identified some possible explanations as to why the results were so different. NMR may be overestimating the solubility by not allowing the most stable fibrils to form, meaning more protein can stay in solution. In the OPA and HPLC-MS experiments, some protein may be lost during the procedure by sticking to surfaces of the equipment used. Finally, OPA may be reporting a higher solubility than HPLC-MS because it cannot tell the difference between intact, unmodified protein and protein which has degraded or been chemically modified, while HPLC-MS only detects the intact protein. As all three methods have different strengths and drawbacks, I concluded that all three should be further investigated.

• author: Stemme, Elin
• supervisor: Sara Linse; Max Lindberg; Jing Hu; Emma Sparr
• organization: Biophysical Chemistry
• course: KFKM05 20241
• year: 2024
• type: H3 - Professional qualifications (4 Years - )
• subject: Technology and Engineering
• keywords: amyloids, solubility, amyloid beta, biotechnology
• language: English
• id: 9165851
• date added to LUP: 2024-09-17 11:58:33
• date last changed: 2024-09-17 11:58:33