Institute of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, PR China.
Dermatology Hospital, Southern Medical University, Guangzhou 510091, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Jan 15;1105:193-202. doi: 10.1016/j.jchromb.2018.12.025. Epub 2018 Dec 24.
The supernatant and precipitate protein fractions from human bronchial smooth muscle cells (HBSMC) were analyzed using 1D SDS-PAGE-LC-MS/MS and the performance of the method was compared with that of nondenaturing 2DE-LC-MS/MS applied to the supernatant fraction, the methods and the results have been reported previously. The 1D gel lanes were cut into pieces of the same size, in order to enable the reproduction of digital 1D images of the individual MS-assigned proteins. When the obtained information on individual HBSMC proteins was compared between the two methods, SDS-PAGE is advantageous in visualizing the quantity differences between differently treated samples, whereas nondenatuing 2DE is advantageous in visualizing protein interactions. SDS-PAGE-MS of the supernatant fraction provided the assignment of 2552 proteins and their percent abundance ranged from 3.5% to 2 × 10%. 2DE-MS of the supernatant fraction provided 4323 proteins with percent abundance ranged from 3.6% to 1 × 10%, suggesting that the step of isoelectric focusing served to raise the sensitivity of the method. The proteins in the precipitate fraction, which could not be analyzed by 2DE-MS, were characterized by the abundance of proteins allocated to "membrane" within the category "Cellular component" of UniProtKB database and especially those allocated to "transmembrane" within the subcategory "membrane." On the other hand, there were about 600 "membrane" proteins which showed more than two-fold higher percent abundance in 2DE-MS than in SDS-PAGE-MS. These results showed that 1D SDS-PAGE and nondenaturing 2DE would provide complementary information on the analysis of proteins and protein interactions in cells.
上清液和沉淀蛋白部分从人支气管平滑肌细胞（HBSMC）进行了分析，使用 1D SDS-PAGE-LC-MS/MS 和方法的性能进行了比较与非变性 2DE-LC-MS/MS 应用于上清液部分，该方法和结果已报道。1D 凝胶条带切成相同大小的块，为了能够重现个别 MS 分配蛋白的数字 1D 图像。当比较两种方法之间的个别 HBSMC 蛋白的信息时，SDS-PAGE 在可视化不同处理样品之间的数量差异方面具有优势，而非变性 2DE 则在可视化蛋白相互作用方面具有优势。上清液部分的 SDS-PAGE-MS 提供了 2552 种蛋白质的分配及其丰度百分比范围为 3.5%至 2×10%。上清液部分的 2DE-MS 提供了 4323 种蛋白质，其丰度百分比范围为 3.6%至 1×10%，表明等电聚焦步骤提高了方法的灵敏度。沉淀部分的蛋白质不能通过 2DE-MS 进行分析，其特征是分配给 UniProtKB 数据库的“膜”类别内的蛋白质的丰度，特别是分配给子类别“膜”内的“跨膜”蛋白质的丰度。另一方面，有大约 600 种“膜”蛋白在 2DE-MS 中的丰度比在 SDS-PAGE-MS 中高两倍以上。这些结果表明，1D SDS-PAGE 和非变性 2DE 将提供有关细胞中蛋白质和蛋白相互作用分析的互补信息。
