| Metadata |
| datasetIdentifier | PASS00743 |
| datasetType | SRM |
| submitter | Craig Mageean <craig.mageean@gmail.com> |
| submitter_organization | University of Liverpool |
| lab_head_full_name | Ian Prior |
| lab_head_email | iprior@liv.ac.uk |
| lab_head_organization | Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, L69 3BX, UK |
| lab_head_country | United Kingdom |
| datasetTag | Ras_abundance |
| datasetTitle | SRM and PSAQ analysis of endogenous Ras isoform abundance in isogenic SW48 cell lines, harbouring various K-Ras mutations. |
| publicReleaseDate | 2015-09-09 00:00:00 |
| finalizedDate | 2015-09-09 07:05:43 |
| summary | Selected reaction monitoring (SRM) and protein standard absolute quantification (PSAQ) were utilised to measure the cellular Ras abundance in a range of isogenic, human colorectal SW48 cell lines, harbouring a range of engineered mutations. H-, K4B- and N-Ras levels were monitored and measured. |
| contributors | Mageean, C.J.,
Griffiths, J.R.,
Smith, D.L.,
Clague, M.J.,
Prior, I.A. |
| publication | Mageean, C.J., Griffiths, J.R., Smith, D.L., Clague, M.J., Prior, I.A. Absolute quantification of endogenous Ras isoform abundance. PLoS ONE, submitted. |
| growth | Full-length, his-tagged, isotope-labelled Ras proteins were expressed in auxotrophic AT713 E.coli and grown in M9 minimal media supplemented with heavy isotopes of arginine and lysine. SW48 cells were grown in unlabelled McCoy's 5A media in 100 mm dishes until ~80 confluent, before trypsinisation, cell counting and lysis. |
| treatment | No treatment was applied to the cell lines prior to lysis. |
| extraction | Full-length, isotope-labelled Ras proteins were extracted from auxotrophic bacteria using lysozyme and sonication, before centrifugation and protein purification using his-tag pulldown. Proteins were further purified with gel filtration. Endogenous protein from isogenic SW48 cells were extracted through lysis, on ice, with RIPA buffer. |
| separation | Proteins were separated using SDS-PAGE. Following in-gel digestion, peptides were separated using 60-minute, linear 8-35% [v/v] ACN gradient in 0.1% [v/v] formic acid at a flow rate of 400 nl/min, with column temperature at 60ēC, using a nanoAcquity (Waters, C18, 5 μm particle size, 180 μm × 20 mm, Symmetry, 2G-V/M trap column and C18, 1.7 μm particle size, 75 μm × 250 mm BEH130 column). |
| digestion | Following SDS-PAGE, a gel band containing the Ras proteins was excised. Proteins were digested through in-gel digestion with trypsin gold (Promega). |
| acquisition | A minimum of 3 transitions per precursor ion were monitored, with most precursors monitored with 4 or 5 transitions. Each transition was validation through the capture of MS/MS spectra of each peptide using IDA or MIDAS and the order of transition intensity was maintained between endogenous and isotope-labelled peptides. |
| informatics | All wiff files were analysed using Skyline (version 2.4) |
| instruments | AB SCIEX 4000 QTRAP |
| species | Human |
| massModifications | K+8.014199, R+10.008269 |
