Development and application of wide-range gradient gel electrophoresis to proteome analysis

Abstract

SDS is widely used to treat proteins that are difficult to solubilize and digest and improve protein separation in SDS-PAGE. However, SDS interferes with subsequent analyses and needs to be removed prior to digestion and LC-MS/MS analysis, whereas the conventional SDS-PAGE lacks the ability to efficiently remove SDS and retain low-molecular-weight proteins and peptides. In the present work, we developed a wide-range gradient gel electrophoresis (WGGE) system in a vertical slab gel electrophoresis cell, which was primarily composed of a 4-20% continuous gradient polyacrylamide gel separation layer and two interception layers with even higher concentrations (30% and 50%, respectively). The main advantages of the system are simultaneously cleaning up SDS-solubilized samples, separating proteins and intercepting low-molecular-weight proteins and peptides, thereby simplifying experimental operation, improving protein recovery and enhancing the total efficiency of proteome analysis. Using this system, about 87.25% of SDS in the sample and gel was electrophoretically removed and a peptide with a molecular weight of 3.75 kDa was efficiently intercepted. Combined with CapLC-MS/MS, the WGGE system was applied to the analysis of rat liver membrane-enriched protein samples and the results indicated that the WGGE-based strategy is suitable for the identification of proteins varying in molecular weight, pI, hydrophobicity, etc., suggesting potential applications in global and comparative analyses of various proteomes.

Hippocampal proteomic changes of susceptibility and resilience to depression or anxiety in a rat model of chronic mild stress

“…The whole proteins were effectively extracted using the SDT lysis buffer. Before proceeding with trypsin digestion and MS detection, a short gel-based approach was employed to eliminate SDS interference 28,39 . In the current study, a total of 2593 distinct proteins were identified and 2566 were quantified with less than 1% FDR (Supplementary Table S2).…”