Presentation Information
[P01-102]Development of quantitative analysis methods for the interior of protein droplets
○Yu Tsuruta1, Yuanjia Lin1, Mai Watabe2,3, Kosuke Ino1, Hitoshi Shiku1, Masaki Okumura2,3, Hiroya Abe1,2 (1. Graduate School of Engineering, Tohoku University (Japan), 2. Frontier Research Institute for Interdisciplinary Sciences, Tohoku University (Japan), 3. Graduate School of Life Sciences, Tohoku University (Japan))
Keywords:
probe-based analysis,liquid-liquid phase separation,electrochemical measurements,micro-pipettes
[Purpose] Liquid-liquid phase separation (LLPS) is the phenomenon in which biomolecules such as proteins and nucleic acids condense to form droplet-like assemblies in cells. These droplets act as reaction sites for chemical reactions and contribute to stress responses; they are believed to be involved in various biological processes, and understanding their properties is becoming increasingly important. However, since LLPS is a reversible and dynamic reaction—a delicate phenomenon whose susceptibility to occurrence varies significantly with even minor differences in pH or molecular concentration—quantitative analytical methods for it are currently very limited. Therefore, in this study, we investigated the development of a high-spatial-resolution quantitative analytical method by combining micro-pipettes and microelectrodes fabricated through microfabrication with the sampling method and electrochemical measurement methods described below. These analytical methods are intended to contribute to the elucidation of the behavior of internal substances and molecular transport within and outside droplets, as well as the redox environment.
[Method] For the LLPS model system, we used P5¹), a protein that forms droplets in response to Ca2+ stimulation. For sampling, a glass probe was used as a microsyringe to aspirate and recover P5 droplet solution. The recovered solution was analyzed by absorbance measurements to determine P5 and Ca2+ concentrations. For electrochemical measurements, a platinum wire was inserted into a probe to fabricate a microelectrode with a tip diameter of approximately 20 μm. Ferrocene methanol (FcCH2OH, 1 mM), a hydrophobic small-molecule model, was added as an electrochemical indicator. The microelectrode was inserted into the droplet solution, and oxidation currents inside and outside the droplets were measured using i–t transient analysis. A platinum microelectrode was used as the working electrode, and an Ag/AgCl electrode served as both the reference and counter electrodes. The i–t transient data were converted into Cottrell plots to calculate diffusion coefficients inside and outside the droplets. The concentration of FcCH2OH was then determined using the steady-state current equation.
[Results and Discussion] Using the sampling method, absorbance measurements confirmed that the protein concentration inside the droplets was higher than the initial concentration prior to droplet formation, while the concentration outside the droplets was lower. This result is believed to be due to the concentration effect caused by LLPS. The results of the electrochemical measurements confirmed that there was a difference of more than 10-fold in the diffusion coefficients of FcCH2OH between the inside and outside of the droplets, indicating that protein concentration affected the diffusion dynamics of FcCH2OH. Meanwhile, the concentration of FcCH2OH showed a trend similar to that observed in the sampling method: it was higher inside the droplets and lower outside, compared to the initial 1 mM concentration.
[Conclusion] Using the two measurement methods developed, we quantitatively obtained information on the concentration and diffusivity of proteins in liquid-liquid phase-separated droplets. These methods were shown to be effective for acquiring localized information within liquid-liquid systems.
[References] 1) Young-Ho Lee et al., Nat. Cell. Biol. 27, 1952-1964 (2025).
[Method] For the LLPS model system, we used P5¹), a protein that forms droplets in response to Ca2+ stimulation. For sampling, a glass probe was used as a microsyringe to aspirate and recover P5 droplet solution. The recovered solution was analyzed by absorbance measurements to determine P5 and Ca2+ concentrations. For electrochemical measurements, a platinum wire was inserted into a probe to fabricate a microelectrode with a tip diameter of approximately 20 μm. Ferrocene methanol (FcCH2OH, 1 mM), a hydrophobic small-molecule model, was added as an electrochemical indicator. The microelectrode was inserted into the droplet solution, and oxidation currents inside and outside the droplets were measured using i–t transient analysis. A platinum microelectrode was used as the working electrode, and an Ag/AgCl electrode served as both the reference and counter electrodes. The i–t transient data were converted into Cottrell plots to calculate diffusion coefficients inside and outside the droplets. The concentration of FcCH2OH was then determined using the steady-state current equation.
[Results and Discussion] Using the sampling method, absorbance measurements confirmed that the protein concentration inside the droplets was higher than the initial concentration prior to droplet formation, while the concentration outside the droplets was lower. This result is believed to be due to the concentration effect caused by LLPS. The results of the electrochemical measurements confirmed that there was a difference of more than 10-fold in the diffusion coefficients of FcCH2OH between the inside and outside of the droplets, indicating that protein concentration affected the diffusion dynamics of FcCH2OH. Meanwhile, the concentration of FcCH2OH showed a trend similar to that observed in the sampling method: it was higher inside the droplets and lower outside, compared to the initial 1 mM concentration.
[Conclusion] Using the two measurement methods developed, we quantitatively obtained information on the concentration and diffusivity of proteins in liquid-liquid phase-separated droplets. These methods were shown to be effective for acquiring localized information within liquid-liquid systems.
[References] 1) Young-Ho Lee et al., Nat. Cell. Biol. 27, 1952-1964 (2025).
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