Membrane proteins (MPs) play diverse important roles for physical interactions, cell communication, molecular transport, and signal transduction. Membrane proteins comprise approximately 25∼35% of the genome in living organisms, but there are difficulties in the analysis at the protein chemical level, in particular due to low abundance and limited solubility. Sequence information on membrane proteins and their complexes would be beneficial to elucidate their function. Proteins were extracted from pooled whole mouse brains, enriched membrane fractions were prepared using either two commercially available kits or 6-aminocaproic acid under denaturing or native conditions followed by gel-based proteomic approaches using blue native (BN-) and SDS-PAGE with subsequent in-gel digestion with several proteases, chymotrypsin, trypsin followed by nano-LC-ESI-MS/MS analysis on LTQ Orbitrap Velos Pro. By combining three different extraction methods and two separation methods, 28.39% of proteins were identified as either “integral” or “anchored/integral” MPs based on UniProtKB database searches. MPs with more than six transmembrane domains (TMDs) were identified more efficiently from BN-PAGE separation although a higher number of proteins was identified from SDS-PAGE separation. Comparative analysis of MPs containing TMDs via gel-based LC-MS/MS using BN-PAGE and SDS-PAGE may be useful to increase the number of identified membrane proteins in brain. All MS data have been deposited in the ProteomeXchange with identifier PXD000311 (http://proteomecentral.proteomexchange.org/dataset/PXD000311).