PDF(Pubmed)
Abstract：
Microfluidic analytical tools play an important role in miniaturizing targeted proteomic assays for improved detection sensitivity, throughput, and automation. Microfluidic isoelectric focusing (IEF) can resolve proteoforms in lysate from low-to-single cell numbers. However, IEF assays often use carrier ampholytes (CAs) to establish a pH gradient for protein separation, presenting limitations like pH instability in the form of cathodic drift (migration of focused proteins toward the cathode). Immobilized pH gradient (IPG) gels reduce cathodic drift by covalently immobilizing the pH buffering components to a matrix. To our knowledge, efforts to implement IPG gels at the microscale have been limited to glass microdevices. To adapt IEF using IPGs to widely used microfluidic device materials, we introduce a polydimethylsiloxane (PDMS)-based microfluidic device and compare the microscale pH gradient stability of IEF established with IPGs, CAs, and a hybrid formulation of IPG gels and CAs (mixed-bed IEF). The PDMS-based IPG microfluidic device (μIPG) resolved analytes differing by 0.1 isoelectric point within a 3.5 mm separation lane over a 20 min focusing duration. During the 20 min duration, we observed markedly different cathodic drift velocities among the three formulations: 60.1 μm/min in CA-IEF, 2.5 μm/min in IPG-IEF (∼24-fold reduction versus CA-IEF), and 1.4 μm/min in mixed-bed IEF (∼43-fold reduction versus CA-IEF). Lastly, mixed-bed IEF in a PDMS device resolved green fluorescent protein (GFP) proteoforms from GFP-expressing human breast cancer cell lysate, thus establishing stability in lysate from complex biospecimens. μIPG is a promising and stable technique for studying proteoforms from small volumes.
摘要：
微流控分析工具在微型化靶向蛋白质组学测定以提高检测灵敏度方面发挥着重要作用。吞吐量，和自动化。微流体等电聚焦（IEF）可以从低到单细胞数量解析裂解物中的蛋白质形式。然而,IEF测定通常使用载体两性电解质（CA）来建立用于蛋白质分离的pH梯度，以阴极漂移（聚焦蛋白向阴极迁移）的形式存在pH不稳定性等限制。固定的pH梯度(IPG)凝胶通过将pH缓冲组分共价固定至基质来减少阴极漂移。据我们所知,在微尺度上实施IPG凝胶的努力仅限于玻璃微器件。为了使使用IPG的IEF适应广泛使用的微流体设备材料，我们介绍了一种基于聚二甲基硅氧烷（PDMS）的微流体装置，并比较了与IPG建立的IEF的微尺度pH梯度稳定性，CA,以及IPG凝胶和CA的混合制剂(混合床IEF)。基于PDMS的IPG微流体装置(μIPG)在20分钟的聚焦持续时间内在3.5mm分离泳道内分辨出相差0.1等电点的分析物。在20分钟的持续时间内，我们观察到三种配方之间明显不同的阴极漂移速度：在CA-IEF中60.1μm/min，在IPG-IEF中2.5μm/min(与CA-IEF相比降低~24倍)，和1.4μm/min的混合床IEF(与CA-IEF相比降低了43倍)。最后,PDMS设备中的混合床IEF可分辨来自表达GFP的人乳腺癌细胞裂解物的绿色荧光蛋白（GFP）蛋白形式，从而建立了从复杂的生物标本裂解物的稳定性。μIPG是一种用于研究小体积蛋白质形式的有前途且稳定的技术。
