| Metadata |
| datasetIdentifier | PASS00690 |
| datasetType | MSMS |
| submitter | Marco Trevisan-Herraz <mtrevisan@cnic.es> |
| submitter_organization | CNIC |
| lab_head_full_name | Jesús Vázquez |
| lab_head_email | jvazquez@cnic.es |
| lab_head_organization | CNIC |
| lab_head_country | Spain |
| datasetTag | VSMCMouse_iTRAQ8plex |
| datasetTitle | Mouse Vascular Smooth Muscle Cell culture labelled with iTRAQ 8-plex. |
| publicReleaseDate | 2015-07-01 00:00:00 |
| finalizedDate | 2016-04-15 04:34:38 |
| summary | Primary vascular smooth muscle cells were isolated from C57BL/6 mice abdominal and thoracic aortas |
| contributors | Elena Bonzon-Kulichenko, Fernando García-Marqués, Marco Trevisan-Herraz, Sara Martínez-Martínez, Nerea Méndez, Juan Miguel Redondo, and Jesús Vázquez |
| publication | unpublished |
| growth | Cultured in Dulbecco’s modified Eagel’s medium (DMEM) with 20% fetal bovine serum. |
| treatment | VSMCs were rinsed thrice with PBS and left starving for 48 hours in serum-free DMEM. Cells were then treated with 1uM of AngII (Sigma-Aldrich) or vehicle in serum-free culture medium for 2, 4, 6, 8 and 10 h. Cells were lysed with RIPA (NaCl 150 mM, 50 mM Tris pH 8,0, 1% NP-40, 0,5% sodium desoxicholate, 0,1% SDS) supplemented with 1 mM DTT, 3 mM EGTA, 1 mM PMSF and protease inhibitors cocktail (Sigma-Aldrich, San Luis; MO, USA) and protein extracts stored at -80ºC until use. |
| extraction | Whole-cell lysates were prepared by lysing cells with RIPA buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 3 mM EGTA and 1% Nonidet P‐40, 0.5% sodium desoxicholate, 0.1%SDS) supplemented with 1 mM DTT, 1 mM PMSF and a cocktail of protease inhibitors (Sigma-Aldrich, San Luis; MO, USA) on ice for 30 min and then boiling in 2× Laemmli buffer. |
| separation | Peptides were separated into six fractions using MCX cartridges. |
| digestion | Trypsin digestion of all protein extracts was performed using a robust protocol described previously (Bonzon-Kulichenko et al. 2011). VSMCs and total lymphocyte mouse lysates were labelled with iTRAQ8-plex and 4-plex, respectively, according to manufacturer’s instructions. |
| acquisition | LC-MS/MS coupled to a Thermo Scientific Q Exactive instrument, using a top20 data-dependent fragmentation method. |
| informatics | |
| instruments | Thermo Scientific Q Exactive |
| species | Mouse |
| massModifications | static: C+57.021464
static: Nterm+304.2054
static: K+304.2054
variable: M+15.9949 |
