Electrophoretically driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes

Abstract

SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.

Detection and identification of huwentoxin-IV interacting proteins by biotin-avidin chemistry combined with mass spectrometry

“…The enrichment of plasma membrane rich in neuromuscular junctions from adult Sprague-Dawley rats was performed according to previously described methods [15,16]. In brief, the center parts of the diaphragm muscles isolated from the rats anesthetized with ether and killed by decapitation were homogenized with a Dounce homogenator in a protease-inhibitor-containing homogenate buffer (250 mM sucrose, 10 mM Hepes, 1.0 mM EDTA, 100 mM PMSF, pH 7.4).…”

Shotgun analysis of membrane proteomes using a novel combinative strategy of solution-based sample preparation coupled with liquid chromatography–tandem mass spectrometry

“…Rat liver cellular membranes-enriched sample was prepared according to the procedure described previously [27,28]. Briefly, rats were killed after being starved for 18-24 h and the livers were excised.…”

Transferrin recognition based on a protein imprinted material prepared by hierarchical imprinting technique

“…Template could be eluted simultaneously. Furthermore, the mixture contained 10 % SDS (w/v) and 10 % HAc (v/v) was applied to removed transferrin, by unfolding the β-sheet of protein and offering the strong hydrophobic interaction with protein [37,38].…”