A droplet microfluidics approach to measuring protein aggregation

蛋白质聚集在生物学和疾病中起着重要作用。例如，各种综合征 所谓的淀粉样蛋白疾病涉及蛋白质自组装成细长的淀粉样蛋白纤维。关键的第一步 在理解这些过程背后的力量是定义哪些蛋白质可以自我关联-更多 特别是，以确定聚集的序列决定因素。这对于当前的方法是不可行的， 其是低通量的并且只能用于研究非常有限数量的序列。此外，委员会认为， 将蛋白质序列的贡献独立于周围细胞环境分离需要 在生物化学定义的条件下进行测量。我们建议开发一种基于液滴微流体的检测方法， 来测量蛋白质聚集。微滴微流体技术是一种产生和操纵皮升- 大小的水性液滴，嵌入氟化油流中，有效地将液滴与一个 另这些液滴可以以高达5000赫兹的速度产生，单个基因可以被 封装在其中，从而能够分析大型库。我们描述了一种观察蛋白质的方法， 在液滴中进行自组装，并对产生易于聚集的肽的基因进行测序。我们将 将这种方法应用于包含阿尔茨海默氏症相关蛋白Tau变体的两个序列文库。如果 如果成功，该系统可以应用于鉴定自缔合的其他序列，或者序列或 在多种疾病相关蛋白中以及在其他疾病相关蛋白中调节淀粉样蛋白形成的辅因子， 各种聚集现象，如液-液相分离。

Protein aggregation plays an important role in biology and disease. For example, a variety of syndromes termed amyloid diseases involve the self-assembly of proteins into elongated amyloid fibers. A critical first step in understanding the forces that underlie these processes is to define which proteins can self-associate – more specifically, to identify the sequence determinants of aggregation. This is not feasible with current methods, which are low-throughput and can only be used to study a very limited number of sequences. Furthermore, isolating the contribution of protein sequence independent from the surrounding cellular milieu requires measurement in biochemically defined conditions. We propose to develop a droplet microfluidics-based assay to measure protein aggregation. Droplet microfluidics is a technique that generates and manipulates picoliter- sized aqueous droplets, embedded in a stream of fluorinated oil that effectively isolates the droplets from one another. These droplets can be generated at a rate of up to 5000 hertz, and individual genes can be encapsulated in them, enabling the analysis of large libraries. We describe an approach to observe protein self-assembly in droplets and to sequence the genes that give rise to aggregation-prone peptides. We will apply this approach to two sequence libraries comprising variants of the Alzheimer’s associated protein, Tau. If successful, this system could be applied to identify other sequences that self-associate, or sequences or cofactors that modulate amyloid formation in a large variety of disease-associated proteins, as well as in other sorts of aggregation phenomena such as liquid-liquid phase separation.