Development and evaluation of an entirely solution-based combinative sample preparation method for membrane proteomics

Abstract The hydrophobic nature of many membrane proteins, especially integral membrane proteins, brings great difficulties to their analysis. To improve the analysis of membrane proteins, an entirely solution-based combinative sample preparation (CSP) method was developed and its application to the shotgun analysis of rat liver membrane proteomes was evaluated in this study. This CSP method comprehensively uses the strong ability of sodium dodecyl sulfate (SDS) to lyse the membranes and solubilize hydrophobic membrane proteins, the high efficiency of the optimized acetone precipitation method in sample cleanup and protein recovery, and the advantages of sodium deoxycholate (SDC) in improving protein solubilization/digestion as well as being compatible with trypsin activity. Compared with two other representative sample preparation methods, the SDC-assisted membrane-lysing method and the tube gel method, the newly established CSP method exhibited superiority in the recovery and identification of hydrophobic peptides, larger peptides, and highly hydrophobic membrane proteins with multiple transmembrane domains. The CSP method has characteristics of easy operation, low cost, and suitability for treating protein samples in various volumes, particularly large volumes, thereby having potential in the analysis of membrane proteomes with mass spectrometry.