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

•Developed an enhanced SDC-assisted digestion method coupled with LC–MS/MS for shotgun membrane proteomics.•Further optimized the SDC concentration for the extraction and solubilization of membrane proteins.•Found the optimal digestion condition of SDC-assisted method.•Identification of membrane proteins particularly IMPs was obviously improved.•The developed method is easy to operate at low cost.

ABSTRACTDespite the biological importance of membrane proteins, their analysis has lagged behind that of soluble proteins and still presents a great challenge mainly because of their highly hydrophobic nature and low abundance. Sodium deoxycholate (SDC)-assisted digestion strategy has been introduced in our previous papers, which cleverly circumvents many of the challenges in shotgun membrane proteomics. However, it is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of 1% SDC. In this study, an enhanced SDC-assisted digestion method (ESDC method) was developed that incorporates the almost strongest ability of SDC with a high concentration (5%) to lyse membrane and extract/solubilize hydrophobic membrane proteins, and then dilution to 1% for more efficient digestion. The comparative study using rat liver membrane-enriched sample showed that, compared with previous SDC-assisted method and the “universal” filter-aided sample preparation (FASP) method, the ESDC method not only increased the identified number of total proteins, membrane proteins, hydrophobic proteins, integral membrane proteins (IMPs) and IMPs with more than 5 transmembrane domains (TMDs) by an average of 10.8%, 13.2%, 17.8%, 17.9% and 52.9%, respectively, but also enhanced the identified number of total peptides and hydrophobic peptides by averagely 12.5% and 14.2%. These results demonstrated that the ESDC method provides a substantial improvement in the recovery and identification of membrane proteins, especially those with high hydrophobicity and multiple TMDs, and thereby displaying more potential for shotgun membrane proteomics.