Hepatocyte-specific BIRC5 (Survivin)-knockout-mouse model, Subcellular fractionation

ABSTRACT: Experimental design For the label-free proteome analysis 17 mice were used composed of 5 individual wildtype mice for each condition before (WTprePHE) and after hepatectomy (WTpostPHE). For the pre-hepatectomy condition four BIRC5-knockout mice (KOprePHE) were analyzed and three individuals after partial hepatectomy (KOpostPHE). For the LC-MS/MS analysis the samples were pre-fractionated by subcellular protein fractionatio. All samples corresponding to a subcellular fraction were analyzed in an individual label-free study. Subcellular protein fractionation The subcellular fractions were generated using the Subcellular Protein Fractionation Kit for Tissue (Thermo Scientific, Bremen, Germany). Sample preparation and tryptic digestion 5ug of protein were loaded on a 18% Tris-glycine acrylamide gel (Anamed Electrophorese, Grosz-Bieberau, Germany). The samples were allowed to run slightly into the gel (15 min, 100 V) and form a single band of approximately 3mm height. The bands were stained with Coomassie, manually cut from the gel and digested with trypsin (Serva Electrophoresis, Heidelberg, Germany) in 10 mM ammonium bicarbonate buffer (pH 7.8) overnight at 37 degrees C. LC-MS/MS Analysis The RPLC-MS/MS analysis was performed using an Ultimate 3000 RSLCnano system (Thermo Scientific, Bremen, Germany) online coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific, Bremen, Germany). The injection volume was 15 ul of the sample, representing an amount of 250 ng peptides.The MS was operated in a data-dependent mode. Full scan MS spectra were acquired at a resolution of 60,000 in the Orbitrap analyzer, while tandem mass spectra of the twenty most abundant peaks were measured in the linear ion trap after peptide fragmentation by collision-induced dissociation (CID).

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Liver

SUBMITTER: Thilo Bracht  

LAB HEAD: Thilo Bracht

PROVIDER: PXD000625 | Pride | 2014-05-22

REPOSITORIES: Pride