| Metadata |
| datasetIdentifier | PASS00920 |
| datasetType | MSMS |
| submitter | Ulrike Kusebauch <ukusebauch@systemsbiology.org> |
| submitter_organization | Institute for Systems Biology |
| lab_head_full_name | Robert Moritz |
| lab_head_email | Robert.Moritz@systemsbiology.org |
| lab_head_organization | Institute for Systems Biology |
| lab_head_country | USA |
| datasetTag | calcification |
| datasetTitle | Luminal Calcification of the Microvasculature in Fetuin-A Deficient Mice Leads to Premature Aging and Death |
| publicReleaseDate | 2019-12-31 00:00:00 |
| finalizedDate | |
| summary | |
| contributors | Marietta Herrmann, Anne Babler, Irina Moshkova, Felix Gremse, Fabian Kiessling, Ulrike Kusebauch, Valentin Nelea, Rafael Kramann, Robert L. Moritz, Marc McKee, Willi Jahnen-Dechent |
| publication | Herrmann, M, Babler, A, Moshkova, I, Gremse, F, Kiessling, F, Kusebauch, U, Nelea, V, Kramann, R, Moritz, RL, McKee, M, Jahnen-Dechent, W, submitted |
| growth | |
| treatment | |
| extraction | Interscapular brown adipose tissue, skin (interscapular region) and heart were dissected. Mineralized lesions were scraped out under a dissection microscope (Leica MZ6). Mineralization-free tissue was collected as control tissue. Samples were frozen in liquid nitrogen and stored at -70 °C. Samples were thawed, transferred to 2 mL reaction tubes, and incubated with SDS sample buffer (0.25 M TRIS, 8.2% SDS, 20% glycerin, 10% bmercaptoethanol, bromophenol blue) containing 40 mM EDTA at 96 °C for 5 min at 10 μl per 1 mg tissue. The supernatant was removed, and tissue pellets were homogenized for 2.5 min at 25 Hz in a mixer mill (Tissue Lyser II, Qiagen, Hilden, Germany). Following this, samples were boiled for 5 minutes. |
| separation | Protein extracts were separated in 12.5% polyacrylamide gels using SDS-PAGE. Gels were washed and proteins were stained with Imperial Protein Stain (Thermo Fisher Scientific, Rockford, IL, USA) for 2 hours. Unbound stain was removed by washing in ultra-pure water over night. Pictures of gels were recorded using a digital camera. Gel lanes were cut into 2-mm bands using a GridCutter (The Gel Company, San Francisco, USA). |
| digestion | Individual gel slices were subjected to in-gel reduction with dithiothreitol (10 mM, 30 min, 56 °C, Merck-Calbiochem, San Diego, USA), alkylation with iodoacetamide (50 mM, 30 min, room temperature, darkness, Fluka, St. Louis, USA) and digestion with 150 ng trypsin (5 robotic liquid handler (Tecan Systems, Inc, San Jose, USA). Peptides were extracted with 50 μl 50% (v/v) acetonitrile, 50 mM ammonium bicarbonate, concentrated by centrifugal evaporation (Savant, Thermo-Fisher Scientific, USA) and re-solubilized in 20 μl 2% (v/v) acetonitrile, 0.1% formic acid, in water. |
| acquisition | Peptides were analyzed on a LTQ Orbitrap Velos with nano electrospray ionization source (Thermo-Fisher Scientific, San Jose, CA) connected to a 1100 Series HPLC (Agilent Technologies, Santa Clara, CA) with an electronically controlled flow splitter for nano flow rates. Peptides were loaded to a trap column packed with ReproSil-Pur C18-AQ (15x0.075 mm I.D., 120 Å, 3 μm, Dr. Maisch, Ammerbuch-Entringen, Germany) for 6 min using 2% acetonitrile, 0.1% formic acid in water at a flow rate of 3 μl/min. Peptide separation was performed with an in-house packed capillary column (150x0.075 mm I.D., 120 Å, 3 μm, Dr. Maisch ReproSil-Pur C18-AQ, Ammerbuch-Entringen, Germany) using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with a gradient from 2% to 35% B in 60 min at a flow rate of 0.3 μl/min. Survey full scan MS spectra were acquired in the mass range m/z 300–1800 in the Orbitrap analyzer at a resolution of 60,000. The five most intense ions in the survey scan were fragmented by collision-induced dissociation in the LTQ. Charge state one, and unassigned charges, were rejected. MS/MS spectra were acquired upon a minimal signal of 500 counts, with an isolation width of two and a normalized CE of 35. Dynamic exclusion was enabled to exclude precursors for 60 s after three observations |
| informatics | Thermo Xcalibur .RAW files were converted to mzML using ProteoWizard msconvert (version 3.0.03495) MS/MS spectra were searched with Comet (version 2015.02 rev 5) against the mouse proteome obtained from UniProt (www.uniprot.org, release 2016_05), common contaminants and a sequence-shuffled decoy counterpart were appended to the database. Peptides were allowed to be semi-tryptic with up to two internal cleavage sites. The search parameters included a fixed modification of +57.021464 to account for carbamidomethylated cysteines and variable modifications of +15.994915 for oxidized methionines and +42.010565 for N-terminal acetylation. The search results were processed with the Trans-Proteomic Pipeline (version 4.8.0 PHILAE) including PeptideProphet, iProphet and ProteinProphet. Peptide spectrum matches (PSMs) generated by the search engine were analyzed with PeptideProphet to assign each PSM a probability of being correct. The accurate mass binning and nonparametric model were used in the PeptideProphet analysis. PeptideProphet results were further processed with iProphet to refine the PSM-level probabilities and compute peptide-level probabilities, and subsequently with ProteinProphet to calculate probabilities for protein identifications. Only proteins identified with a probability of 0.9 in each sample corresponding to an error of 1% were considered in this analysis. |
| instruments | LTQ Orbitrap Velos |
| species | Mouse |
| massModifications | static: C+57.021464, variable: M+15.994915, N-term+42.010565 |
