| Metadata |
| datasetIdentifier | PASS00430 |
| datasetType | MSMS |
| submitter | Matthew Monroe <matthew.monroe@pnnl.gov> |
| submitter_organization | Pacific Northwest National Laboratory |
| lab_head_full_name | Richard D. Smith |
| lab_head_email | dick.smith@pnnl.gov |
| lab_head_organization | Biological Sciences Division |
| lab_head_country | United States |
| datasetTag | SysVirol_SCL005 |
| datasetTitle | Systems Virology SCL005: icSARS CoV (wild type and delta ORF 6 mutant)- and mock-infected 2B4 cell time course |
| publicReleaseDate | 2014-02-08 00:00:00 |
| finalizedDate | 2014-03-19 11:54:21 |
| summary | This submission corresponds to peptides identified from proteomics analysis of tryptic digests of 2B4 cells infected with SARS (icSARS CoV and icSARS CoV delta ORF 6 mutant) virus. Cells were mock-infected or infected with virus (n = 3 each) at an MOI of 5 and harvested at 0, 3, 7, 12, 24, 30, 36, 48, 54, 60, and 72 h post-infection. Each sample was subjected to tryptic digestion and analysis by capillary LC-MS (Exactive MS) using the PNNL-developed accurate mass and time tag approach. More information can be found at the following web site: https://www.systemsvirology.org/project/home/begin.view? |
| contributors | Tom Metz |
| publication | unpublished |
| growth | Infections for proteomics analyses were performed in a clonal population of Calu3-2B4 cells expressing high levels of the SARS-CoV cellular receptor angiotensin converting enzyme 2 (ACE2, human lung adenocarcinoma cells). Calu3-2B4 cells were grown in minimal essential media (MEM; Invitrogen-Gibco) containing 20% fetal bovine serum (HyClone) and 1% antibiotic-antimycotic mixture (Invitrogen-Gibco).
Viral titration assays were performed in Vero E6 cells. Vero E6 cells were maintained in MEM (Invitrogen-Gibco) containing 10% fetal clone II (HyClone) and 1% antibiotic-antimycotic (Invitrogen-Gibco). Wild-type SARS-CoV (icSARS-CoV) was derived from the Baric laboratory’s infectious clone constructs. Briefly, genome fragments were amplified in Escherichia coli, excised from plasmids by restriction digestion, ligated, and purified prior to in vitro transcription reactions to synthesize full-length genomic RNA, which was transfected into Vero E6 cells. The media from transfected cells were harvested and served as seed stocks for subsequent experiments. Viral genomes were confirmed by sequence analyses prior to use in any experiments. All work was performed in a biosafety level 3 facility supported by redundant fans. |
| treatment | To determine the pattern of differential protein expression for ic-SARS-CoV-infected and mock-infected cells, Calu3-2B4 cells were plated in triplicate under each condition at each time point, washed prior to infection, infected at a multiplicity of infection of 5 (MOI 5), and incubated at 37°C for 40 min. The inoculum was removed, cells were washed 3 times with 1x phosphate-buffered saline (PBS), and then fresh medium was added prior to time zero. Medium was collected to determine viral titers at each time point (0, 3, 7, 12, 24, 30, 36, 48, 54, 60, and 72 h post-infection) for each well. |
| extraction | Cells were washed 3 times in cold 150mMammonium bicarbonate buffer, lysed for 15 min in 8 M urea, and stored at 80°C. |
| separation | |
| digestion | Lysate protein concentrations were determined by BCA protein assay and diluted to a uniform final volume in 50 mM ammonium bicarbonate pH 7.8. Proteins were reduced with 10 mM dithiothreitol, followed by alkylation of free sulfhydryl groups with 40 mM iodoacetamide at 37°C in the dark; each reaction was performed for 1 h at 37°C with constant shaking at 800 rpm. Denatured and reduced samples were diluted 10-fold with 50 mM ammonium bicarbonate pH 7.8, and CaCl2 was added to a final concentration of 1 mM prior to enzymatic digestion. Sequencing-grade modified trypsin was activated by adding 20 μL of 50 mM ammonium bicarbonate pH 7.8 to 20 μg lyophilized trypsin and incubating for 10 min at 37˚C. Activated trypsin was then added to the samples at 1:50 (w/w) trypsin-to-protein ratio, and samples were digested at 37˚C for 3 h with constant shaking at 800 rpm; reactions were quenched by rapid freezing in liquid nitrogen. Digested samples were desalted using solid phase extraction columns (Discovery C18, Supelco, Bellefonte, PA) according to the manufacturer’s instructions. Samples were then concentrated to 100 μL in vacuo (Speed-Vac SC 250 Express, Thermo Savant, Holbrook, NY), and a BCA protein assay was performed to verify final peptide concentrations. Samples were stored at –80˚C until quantitative LC-MS analyses. |
| acquisition | Dried peptide samples were reconstituted in 30 µL of 25 mM ammonium bicarbonate, pH 7.8 and analyzed in triplicate and random order using a 4-column custom-built capillary LC system coupled online to an Exactive mass spectrometer (Thermo Scientific) by way of an in-house manufactured electrospray ionization interface. The temperature of the heated capillary and the ESI voltage were 250°C and 2.2 kV, respectively. Data were collected over the mass range 400-2,000 m/z. |
| informatics | Quantitative LC-MS datasets were processed via the PNNL-developed accurate mass and time (AMT) tag approach using the PRISM Data Analysis system, which is a series of software tools developed in-house (e.g. Decon2LS and VIPER; freely available at http://ncrr.pnl.gov/software/). Individual steps in this data processing approach are reviewed here (PMID: 16429408). The peptide identities of detected features in each dataset (i.e. a single LC-MS analysis) were determined by comparing their measured monoisotopic masses and NETs to the calculated monoisotopic masses and observed NETs of each of the 56,220 peptides contained in a filtered Calu-3 peptide AMT tag database within initial search tolerances of ± 6 ppm and ± 0.025 NET for monoisotopic mass and elution time, respectively. The peptides identified from this matching process were retained as a matrix for subsequent data analysis. |
| instruments | Thermo Scientific Exactive |
| species | Human |
| massModifications | none |
