| Metadata |
| datasetIdentifier | PASS00475 |
| datasetType | SRM |
| submitter | Sofia Waldemarson <sofia.waldemarson@immun.lth.se> |
| submitter_organization | Lund University |
| lab_head_full_name | Peter James |
| lab_head_email | peter.james@immun.lth.se |
| lab_head_organization | Immunotechnology, Lund University |
| lab_head_country | Sweden |
| datasetTag | BRCA_SRMclass |
| datasetTitle | Proteomic analysis of breast tumors follows the mRNA intrinsic molecular subtypes with different classifiers |
| publicReleaseDate | 2014-04-17 00:00:00 |
| finalizedDate | 2014-04-10 05:51:17 |
| summary | The data describes an SRM assay that was used for subtyping breast cancer. Results were compared to RNA expression analysis data and we found the protein classification to be highly similar to the intrinsic subtypes based on gene expression. |
| contributors | Sofia Waldemarson, Morten Krogh, Emila Kurbasic, Paolo Cifani, Tord Berggård, Åke Borg, Peter James |
| publication | submitted |
| growth | These are fresh frozen human tumor samples. |
| treatment | Fresh, resected tumor material were snap frozen and homogenized while frozen. |
| extraction | The powderized tumor material was dissolved in a lysis buffer containing 8M urea, 30 mM Tris, 5 mM magnesium acetate and 4% CHAPS, pH 8.5. The samples were kept on ice for 20 min and repeatedly vortexted. The samples were spun down and the protein concentration of the supernatant was determined. |
| separation | Samples were run into SDS-PAGE gels but not allowed to separate. Each sample was cut out and placed in Eppendorf tubes. |
| digestion | The coomassie blue color was washed away with 50 mM Ammonium bicarbonate (Ambic). The proteins were reduced in 10mM Dithiotreitol in 100mM Ambic and alkylated using 55mM Iodoacetamide in 100mM Ambic. Samples were cleaned using UltraMicroSpin Columns, 3-30 μg capacity (The Nest Group). |
| acquisition | The SRM measurements were performed on a TSQ Vantage triple stage quadrupole mass spectrometer (Thermo, Bremen, Germany) equipped with a nano-electrospray ion source (Thermo Electron) and coupled to an Eksigent 2D NanoLC system (Eksigent technologies, Dublin, CA, USA). The peptides were eluted on analytical columns packed in-housed in 0.075 mm id, 100 mm PicoFrits (New Objective, Woburn, MA) with 3 μm ReproSil C18-AQ particles (Dr. Maisch, Germany) to a length of 12 cm with a gradient of 97% solvent A at 0-5 min, 85% A at 8 min, 65% A at 42 min, 10% A at 45-50 min. 41 tumor samples were analyzed, each in duplicate. |
| informatics | Raw-data files were converted into mzML through ProteoWizzard and imported into Skyline software for manual data analysis to good quality chromatograms. All files accepted for further analysis was integrated using the Anubis automated software for SRM analysis. |
| instruments | Thermo Scientific TSQ Vantage |
| species | Human |
| massModifications | C+57.021464 |
