1D-SDS-PAGE and Nano-LC-MS/MS for Membrane Proteomics of Mouse Liver Microsomes (MPI sample) and its Application to Human Proteomics of ER from Jurkat Cells

Abstract

Proteomic profiling of mouse liver microsomes was performed by SDSpolyacrylamide gel electrophoresis (1D-SDS-PAGE) and nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) for the pilot study of Membrane Proteome Initiative (MPI) of AOHUPO.More than one hundred proteins were identified with high MS/MS search score, and most of those proteins have been reported to be membrane associated proteins such as microsomal glutathione S-transferase 1, cytochrome P-450 and low density lipoprotein receptor-related proteins. This platform was used for ER membrane proteomics of Jurkat cells of a human lymphoblatic lymphoma cell line to find marker proteins for heat stress. The microsomes were prepared by two-step centrifugation of liver homogenate, firstly the homogenate was centrifuged at 15,000 g for 15 min to yield supernatant and secondly the supernatant was centrifuged at 132,000 g for 60 min to yield microsomes pellet. The microsome pellet was suspended in 0.1 M sodium carbonate solution containing 0.5 mM PMSF and 10 ug/mL of aprotinin and leupeptin to yield highly purified microsomes in the pellet by centrifugation at 132,000 g for 60 min. The purified microsomes were pretreated with ice cold acetone-methanol (8:1) solution (Anal.Biochem., 273, 313-315, 1999) to be applied for the separation of membrane proteins by 1D-SDS-PAGE with a gradient gel (5-20%) to yield the fine separation of proteins in more than 50 bands which were visualized by Coomassie Blue staining. The protein bands were cut out and submitted to in gel doi:10.4172/jpb.s1000091