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

Enhanced SDC-assisted digestion coupled with lipid chromatography-tandem mass spectrometry for shotgun analysis of membrane proteome

“…Preparation of rat liver membrane-enriched fraction was performed according to the previous paper [21]. All the experiments were approved by the Committee on the Use and Care of Animals at the Hunan Province, P. R. China and were conducted in accordance with the guidelines established by the Committee.…”

Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics

“…This is predominantly due to their amphipathic quality, making membrane proteins extremely difficult to adequately extract, enrich, and solubilize from their native environment. 2,3,9–13 In addition to their amphipathic nature, membrane proteins are present in low levels, 1,4,5,13,14 increasing the difficulty of their detection in cell lysates. Despite the difficulties involved in their investigation, their important roles in biological processes and as drug targets make them an important focus of proteomic analyses.…”

“…1,7,14,21 Also, the detergent must be removed prior to analysis because it can affect chromatographic separation and suppress ionization in the MS. 5,10,12,20,26,27 The acid-labile surfactant RapiGest SF (Waters) has been shown to greatly enhance membrane protein solubility 3 and allow 100% enzyme digestion at surfactant concentrations of 0.1% and can be easily removed prior to MS analysis via acidification. 29 The acid-insoluble detergent sodium deoxycholate (SDC) has also been reported to improve protein solubility and retain trypsin digestion efficiency at extremely high concentrations 9,10,21,30 (with 10% SDC, trypsin retained 77.4% activity), 31 and SDC can be removed prior to MS analysis via acidification or by ethyl acetate phase transfer. 4,30,32 Many other common surfactants, such as sodium dodecyl sulfate (SDS), can be utilized; however, most require thorough removal via dialysis or chromatography techniques, which can be problematic for high-throughput analyses and low abundance proteins.…”

Sample preparation for the analysis of membrane proteomes by mass spectrometry

“…In particular, when the method is applied to the cleanup of SDS-solubilized membrane proteome samples, a series of problems exists, for example, how to precipitate/recover proteins and remove SDS efficiently, how to re-dissolve and digest the precipitated proteins with high efficiency, etc. In the study of Lin et al (2012), the investigators optimized the experimental conditions for the application of acetone precipitation method to the cleanup of SDS-solubilized membrane protein samples, used enzyme-and MS-compatible SDC to overcome the problems of re-dissolution and digestion of acetone precipitated proteins, and thus developed an entirely solution-based combinative strategy that comprehensively utilizes the advantages of selected detergents and the optimized sample cleanup method to efficiently improve the analysis of membrane proteomes. This strategy overcomes some inherent limitations of the conventional sample preparation methods and is easily operated at low cost and suitable for the analysis of membrane proteomes varying in type and sample volume, etc.…”