Due to their extremely hydrophobic
nature, the analysis of integral
membrane proteins (IMPs) is of great challenge. Although various additives
have been applied to improve the solubility of IMPs, they still suffer
from low solubilization efficiency, incompatibility with trypsin digestion,
or interference with MS detection. Herein, the systematic study on
the effect of ionic liquid structure on membrane protein solubilization
and trypsin biocompatibility was performed, based on which 1-dodecyl-3-methylimidazolium
chloride (C12Im-Cl) was selected for the sample preparation of IMPs.
Compared with other commonly used additives, such as sodium dodecyl
sulfate (SDS), Rapigest, and methanol, C12Im-Cl showed the best performance.
In addition, with a strong cation exchange trap column, it could be
easily removed after trypsin digestion, which not only was beneficial
to avoid protein precipitation during digestion but also had no adverse
effect on LC-MS-based separation and detection. Such a C12Im-Cl-assisted
sample preparation method was further applied to the membrane proteome
analysis of rat brain. Compared with the SDS-assisted method, 1.4
and 3.5 times improvement on the identified IMP and hydrophobic peptide
number were achieved (251 vs 178, and 982 vs 279). All these results
demonstrated that the C12Im-Cl-assisted sample preparation method
is of great promise to promote the large-scale membrane proteome profiling.