| Metadata |
| datasetIdentifier | PASS00653 |
| datasetType | SRM |
| submitter | Thilo Bracht <thilo.bracht@rub.de> |
| submitter_organization | Ruhr-Universität Bochum |
| lab_head_full_name | Thilo Bracht |
| lab_head_email | thilo.bracht@rub.de |
| lab_head_organization | Ruhr-Universität Bochum |
| lab_head_country | Germany |
| datasetTag | HepaticFibrosis |
| datasetTitle | Quantification of 7 Disease-associated Proteins in Different Stages of Hepatic Fibrosis |
| publicReleaseDate | 2015-03-26 00:00:00 |
| finalizedDate | 2015-03-26 06:27:37 |
| summary | We performed a targeted proteomics experiment (MRM/SRM) to verify the disease-associated expression of seven proteins in a cohort of patients suffering of different stages of hepatic fibrosis (n=68, stages F1-F4). |
| contributors | Thilo Bracht, Vincent Schweinsberg, Don Marvin Voß, Wael Naboulsi, Barbara Sitek |
| publication | Thilo Bracht, Vincent Schweinsberg, Martin Trippler, Michael Kohl, Maike Ahrens, Juliet Padden, Wael Naboulsi, Katalin Barkovits, Dominik A. Megger, Martin Eisenacher, Christoph H. Borchers, Jörg F. Schlaak, Andreas-Claudius Hoffmann, Frank Weber, Hideo A. Baba, Helmut E. Meyer and Barbara Sitek, Analysis of disease-associated protein expression using quantitative proteomics - Fibulin-5 is expressed in association with hepatic fibrosis, Journal of Proteome Research, PMID 25807371 |
| growth | None |
| treatment | None |
| extraction | Biopsied specimens were lysed and homogenized in 40 µL of sample buffer (30 mM Tris, 7 M Urea, 2 M Thiourea, 0,1% SDS, pH 8,5). The samples were sonicated and insoluble compounds were subsequently removed by centrifugation (15000 g for 10 min). The protein concentration of supernatants was determined using the Bradford assay (Biorad, Hercules, CA, USA). |
| separation | 15 µg of protein per sample were loaded on an 18% Tris-glycine polyacrylamide gel (Anamed Elektrophorese, Groß-Bieberau, Germany) and allowed to run slightly into the gels (15 min, 100 V). The protein bands were stained with Coomassie and manually cut from the gels. |
| digestion | Digestion of proteins was performed overnight at 37 °C with trypsin (Serva Electrophoresis, Heidelberg, Germany) in 10mM ammonium bicarbonate buffer (pH 7.8). The peptides were extracted from the bands with 20 μL of 50% acetonitrile in 0.1% FA (1:1) by sonication (15 min, on ice) for two times. Subsequently, the peptides were vacuum dried, dissolved in 0.1% FA and the peptide concentration was determined by amino acid analysis. |
| acquisition | MRM experiments were conducted using an Agilent 6490 triple quadrupole MS (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 1290 Infinity Binary HPLC standard-flow system (Agilent Technologies, Santa Clara, CA, USA). A sample volume of 20 µL was injected and separated with a flow rate of 0.4 mL/min on an analytical C18 column (Agilent ZORBAX Eclipse Plus Rapid Resolution HD, 2.1x150 mm, 1.8 µ, column temperature 50 °C). Peptides were separated using a multi-step gradient from 97 % solvent A (0.1% FA) and 3 % solvent B (84% ACN, 0.1 % FA) to 90 % solvent B (time: % B): 0 min: 3% B; 2 min: 11% B; 15 min: 19% B; 20 min: 29% B; 22 min: 39% B; 25 min: 45% B; 27 min: 90% B; 29 min: 90% B; 30min: 3% B. After every sample the complete gradient was run as a washing step to exclude carry-over between individual samples. Peptides were ionized by a standard-flow ESI source (Agilent Jet Stream) using the following parameters: 3000 V capillary voltage, 300 V nozzle voltage, 11 L/min sheath gas flow at a temperature of 250 °C, 15 L/min drying gas flow at a temperature of 150 °C and 30 psi nebulizer gas flow. The QQQ-MS was operated using the mass hunter workstation (ver. B.06.00 Service Pack 1). The settings were 380 V default fragmentor voltage and a 5 V cell accelerator potential, 541 V peptide-specific collision energy (CE), and unit resolution (0.7 Da full-width-at-half- maximum, FWHM) in the first and third quadrupole. The cycle time given to monitor all transitions was 500 ms with a maximum number of 18 transitions monitored in parallel with a dwell time of a minimum of 26 ms and a maximum of 82 ms. |
| informatics | Data was analyzed using skyline (Skyline ver. 2.5.0.6157). Ratios between the stable isotope-labeled peptides and the endogeneous peptides were used for relative quantification. |
| instruments | Agilent 6490 |
| species | Human |
| massModifications | K+8.014198
R+10.008268
F+10.027228 |
