Comparison of the performance of 1D SDS-PAGE with nondenaturing 2DE on the analysis of proteins from human bronchial smooth muscle cells using quantitative LC-MS/MS

Abstract

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 × 10−4%. 2DE-MS of the supernatant fraction provided 4323 proteins with percent abundance ranged from 3.6% to 1 × 10−5%, 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.