The UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, Berkeley, California 94720, United States.
Department of Bioengineering, University of California, Berkeley, California 94720, United States.
Anal Chem. 2024 May 28;96(21):8648-8656. doi: 10.1021/acs.analchem.4c00788. Epub 2024 May 8.
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.
微流控分析工具在将靶向蛋白质组学分析微型化方面发挥着重要作用，可提高检测灵敏度、通量和自动化程度。微流控等电聚焦 (IEF) 可以从低至单细胞数量的裂解物中分辨蛋白质形式。然而，IEF 分析通常使用载体两性电解质 (CA) 来建立 pH 梯度以进行蛋白质分离，存在一些局限性，例如 pH 不稳定，表现为阴极漂移（聚焦蛋白质向阴极迁移）。固定化 pH 梯度 (IPG) 凝胶通过将 pH 缓冲成分共价固定到基质上来减少阴极漂移。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。为了将使用 IPG 的 IEF 适配到广泛使用的微流控器件材料，我们引入了一种基于聚二甲基硅氧烷 (PDMS) 的微流控器件，并比较了使用 IPG、CA 和 IPG 凝胶和 CA 的混合配方（混合床 IEF）建立的微尺度 IEF 的 pH 梯度稳定性。基于 PDMS 的 IPG 微流控器件 (μIPG) 在 20 分钟聚焦时间内，在 3.5 毫米分离通道内分辨出等电点相差 0.1 的分析物。在 20 分钟的时间内，我们观察到三种配方之间阴极漂移速度有明显差异：CA-IEF 为 60.1 μm/min，IPG-IEF 为 2.5 μm/min（相对于 CA-IEF 减少约 24 倍），混合床 IEF 为 1.4 μm/min（相对于 CA-IEF 减少约 43 倍）。最后，在 PDMS 器件中的混合床 IEF 从 GFP 表达的人乳腺癌细胞裂解物中分辨出绿色荧光蛋白 (GFP) 蛋白质形式，从而在复杂生物样本的裂解物中建立了稳定性。μIPG 是一种很有前途且稳定的技术，可用于从小体积中研究蛋白质形式。
