| Metadata |
| datasetIdentifier | PASS00450 |
| datasetType | SRM |
| submitter | Vineet Sangar <vsangar@systemsbiology.org> |
| submitter_organization | Institute for Systems Biology |
| lab_head_full_name | Dr Nathan D Price |
| lab_head_email | nprice@systemsbiology.org |
| lab_head_organization | Institute for Systems Biology |
| lab_head_country | USA |
| datasetTag | Secretome_GBM |
| datasetTitle | Quantitative proteomic analysis reveals effects of EGFR, EGFRvIII, PTEN on invasion-promoting proteins secreted by glioblastoma cells |
| publicReleaseDate | 2014-03-20 00:00:00 |
| finalizedDate | 2014-07-08 10:56:38 |
| summary | In this project, we investigated the influence of EGFR pathway alterations on the 65 invasion promoting proteins in GBM secretome. We measured the rationally selected proteins through selected reaction monitoring (SRM) targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII and/or PTEN are expressed in cell lines. |
| contributors | Vineet Sangar, Nathan D Price |
| publication | unpublished |
| growth | |
| treatment | |
| extraction | Secretome was spun down to remove the dead cell and the cell debris. For protein precipitation, 3 volumes of ice-cold acetone were added to the supernatant, vortexed and stored at -20°C overnight. Next day, the mixture was centrifuged at 10,000 rpm for 10 minutes. The resulting precipitate was dissolved in fresh 6M urea/2M thiourea solution and stored at -80°C. |
| separation | |
| digestion | 2.5 μg of protein was dissolved in 6M urea/2M thiouera, reduced in 5 mM dithiothreitol for 45 min at 60°C, alkylated in 15 mM iodoacetamide for 30 min at room temperature in the dark, diluted 10-fold with 25 mM ammonium bicarbonate, and then digested using sequencing grade trypsin (Promega, Madison, WI) at a ratio of 1:30 by weight at 37°C overnight. Next day, the sample was acidified with acetic acid to 10% stop the proteolytic reaction. Digested sample was combined with 100 fmoles of heavy labeled peptides. The samples spiked with standard peptides were desalted on a PEPCLEAN C18 Spin column (Thermo Scientific Pierce) and injected for SRM analysis. |
| acquisition | All SRM analyses were performed on a triple quadrupole (QQQ) mass spectrometer with a ChipCube nanoelectrospray ionization source (Model G6490A, Agilent Technologies, Santa Clara, CA) utilizing a large capacity HPLC chip (G4240–62010, 160 nL trap, 150 mm C18 column, Agilent Technologies, Santa Clara, CA). The mass spectrometer was connected to a 1200 nanoFlow HPLC and 1260 autosampler (Agilent Technologies, Santa Clara, CA). The samples consisting of the tryptic digest and peptide standard were analyzed by a 60-min gradient with a 0.66% per minute ACN slope in the presence of 0.1% FA. Spray voltage was set between 1750V-1900V. Dynamic SRM was performed with a ±4.5 min retention time. The duty cycle was set to 2500 ms and the minimum dwell time for each transition was set to 10 ms. |
| informatics | The protein expression levels (light to heavy ratio; L/H) were calculated with the publicly available SRM analysis program ‘Skyline’ v1.3. After using the Savitzky-Golay method for peak integration, the following criteria were applied for quantifying the peptide: i) peaks for all transitions of a peptide had identical retention times, ii) out of the three peptides, the peptide with the least coefficient of variation was used to quantify the protein levels; iii) peptides with L/H ratio < 0.03 were classified as undetected to remove any variation introduced to peak areas by manual selection of small peaks. Peak areas were calculated by adding the peak areas for all five transitions per peptide. |
| instruments | Agilent 6490A |
| species | Human |
| massModifications | C+57.021464, variable: K+8.014199, R+10.008269 |
