Enhancing Membrane Protein Identification Using A Simplified Centrifugation And Detergent-Based Membrane Extraction Approach

Membrane proteins may act as transporters,receptors,enzymes and adhesion-anchors.They encompass a wide variety of functions required for normal development and physiology,including signal transduction,subcellular compartmentalization,membrane trafficking,protein secretion and their roles in providing and maintaining membrane integrity,accounting for nearly 70%of pharmaceutical drug targets.Efficient enrichment and solubilization of membrane proteins are still major difficulties due to their relatively low abundance and poor solubility.A simplified membrane protein extraction approach combining high-speed centrifugation and detergent along with LC-MS/MS has advantages of time-saving sample processing procedures,good repeatability and effectiveness,thus being developed in the current research for improving enrichment and identification of membrane proteins.Membrane fraction and total cell lysate prepared from Neuro-2A cells were utilized to evaluate the efficiency of current membrane protein extraction method.The same method was also applied to rat brain cortex tissues,and membrane proteins preparation based on Percoll density gradient according to a previously described method was also performed for comparison.It seemed that although centrifugal force was reduced from 900000 x g to 35000× g,we still successfully identified high proportion of membrane proteins,integral proteins and transmembrane proteins in membrane fraction.The method was also able to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains,and provided for improved identification of membrane proteins via improving sequence coverage.This user-friendly method is suitable for wide utilization in the research institutions without an ultra-high speed centrifuge.