Battle Katrina N, Jackson Joshua M, Witek Małgorzata A, Hupert Mateusz L, Hunsucker Sally A, Armistead Paul M, Soper Steven A
Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70803-1804, USA.
Analyst. 2014 Mar 21;139(6):1355-63. doi: 10.1039/c3an02400h.
We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.
我们展示了一种新型微流控固相萃取（μSPE）装置，用于从全细胞裂解物中亲和富集生物素化的膜蛋白。该装置具有一些特性，可应对目前从全细胞裂解物中提取和纯化膜蛋白所面临的挑战，包括能够从提取表面释放富集的膜蛋白组分，以便其可用于下游处理。萃取床采用热压印法在聚甲基丙烯酸甲酯（PMMA）中制造，由3600个微柱组成。通过紫外线/臭氧（UV/O3）处理对PMMA微柱进行活化，可产生表面受限的羧酸基团，并将中性抗生物素蛋白共价连接到μSPE装置表面，该表面用于直接从全细胞裂解物中亲和选择生物素化的MCF-7膜蛋白。生物素部分中包含二硫键连接子，可通过二硫苏糖醇（DTT）孵育释放分离的膜蛋白。膜蛋白的极低水平（约20飞摩尔）能够以约89%的效率被分离和回收，床容量为1.7皮摩尔。蛋白质免疫印迹法表明，与使用基于商业去污剂的方法时存在的显著污染相比，膜蛋白组分中没有胞质蛋白的痕迹。我们强调了未来提高萃取效率和增加μSPE装置动态范围的途径，即利用不同微柱几何形状的计算模拟来指导未来的装置设计。
