MassIVE MSV000096911

Description

Primary human hepatic stellate cells were transduced with lentivirus expressing ABHD17B-FLAG or GFP-FLAG. Cells were lysed, and immunoprecipitation was performed with M2 anti-FLAG beads. Peptides were eluted with FLAG peptide and separated by gel electrophoresis prior to analysis by LC-MS/MS Samples were analyzed at the Taplin Biological Mass Spectrometry Facility at Harvard Medical School using standard protocols for protein sequence analysis by LC-MS/MS. Excised gel bands were cut into approximately 1 mm3 pieces. Gel pieces were then subjected to a modified in-gel trypsin digestion procedure. Gel pieces were washed and dehydrated with acetonitrile for 10 min. followed by removal of acetonitrile. Pieces were then completely dried in a speed-vac. Rehydration of the gel pieces was with 50 mM ammonium bicarbonate solution containing 12.5 ng/ul modified sequencing-grade trypsin (Promega, Madison, WI) at 4C. After 45 min., the excess trypsin solution was removed and replaced with 50 mM ammonium bicarbonate solution to just cover the gel pieces. Samples were then placed in a 37C room overnight. Peptides were later extracted by removing the ammonium bicarbonate solution, followed by one wash with a solution containing 50% acetonitrile and 1% formic acid. The extracts were then dried in a speed-vac (~1 hr). The samples were then stored at 4C until analysis. On the day of analysis the samples were reconstituted in 5 - 10 ul of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nano-scale reverse-phase HPLC capillary column was created by packing 2.6 um C18 spherical silica beads into a fused silica capillary (100 um inner diameter x ~30 cm length) with a flame-drawn tip. After equilibrating the column each sample was loaded via auto sampler (LC Packings, San Francisco CA) onto the column. A gradient was formed and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). As peptides eluted they were subjected to electrospray ionization and then entered into a Velos Orbitrap Pro ion-trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program, Sequest (Thermo Fisher Scientific, Waltham, MA). All databases include a reversed version of all the sequences and the data was filtered to between a one and two percent peptide false discovery rate. [doi:10.25345/C5DF6KF8Q] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: abhd17b ; hepatic stellate cell ; liver fibrosis ; DatasetType:Proteomics

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