Analysis of mouse hippocampus membrane proteome by gel-based LC/MS

Mouse hippocampus membrane fractions were prepared using sucrose density gradient ultracentrifugation. Membrane proteins were solubilized using three different condition; 6-ACA/Triton X-100 or ProteoExtract Native Membrane Protein Extraction Kit (Calbiochem, Cat. No. 444810) or ProteoExtract Transmembrane Protein Extraction Kit (Novagen, Cat. No. 71772-3). Solubilized membrane proteins were separated on Blue Native PAGE or SDS-PAGE. Protein bands were excised, destained for peptide sample preparation. Proteins were reduced (DTT), alkylated (iodoacetamide), and in-gel digested with chymotrypsin or trypsin. Digested peptides were extracted then subjected to nanoHPLC and tandem MS analysis with Thermo Orbitrap Velos Pro mass spectrometer.