Identification Of Mouse Liver And Rat Hippocampal Plasma Membrane Proteins By Liquid Chromatography-tandem Mass Spectrometry

Multidimensional liquid chromatography coupled with tandem massspectrometry was used to identify mouse liver plasma membrane(PM) proteins. A strong cation-exchange(SCX) column worked as the first phase and a reversed-phase capillary column (150 mm 75 m)as the second phase. A pre-column was incorporated between the two phases of chromatography .The mouse liver plasma membrane was obtained by the density-gradient centrifugation and the PM proteins were extracted by detergents-assisted solution. After an optimized tryptic digestion of the PM proteins , the digested peptide mixture was loaded onto the SCX column (5 mm 320 m) . 10 different concentrations of ammonium acetate solution were injected to elute the ion-exchanged peptides respectively .Following desalinisation the eluted peptides were further purified by reversed-phase capillary column(150 mm 75 m) and online analysed by the coupled tandem mass spectrometry. The acquired MS and MS/MS spectra , after being processed with analytical software Proteinlynx ,were utilized to search the Swiss-Prot database with Mascot search engine for protein identification .By this method , a total of 126 proteins were identified ,of which 43 were known membrane proteins .14 of the membrane proteins were integral proteins or proteins with multiple transmembrane domains (TMDs), which were unidentified by using two-dimensional polyacrylamide gel electrophoresis followed bymass spectrometry (2DE-MS). This work provides fundamental information of the mouse liver plasma membrane proteome .We have also made a preliminary study on the proteome of adult rat hippocampus plasma membrane. The hippocampal plasma membrane isolated from the brains of adult rat with sucrose density-gradient centrifugation. The plasma membrane protein were extracted and subjected to one-dimensional (IDE) and two-dimensional gel electrophoresis, followed by in-gel tryptic digestion and analysis through liquid chromatography coupled with tandem mass spectrometry. We Compared the results of the two methods for proteomic analysis of adult rat hippocampus plasma membrane. In the present study, 51 proteins were identified, of which 19 proteins were known plasma membrane proteins, by IDE in combination with liquid chromatography coupled with tandem mass spectrometry. But only one of 19 identified proteins was plasma membrane protein by 2-DE-LC-MS/MS. It appears that 1DE-LC-MS/MS is more suitable for separating and analyzing plasma membrane proteins than 2DE/LC-MS/MS.