PNNL_EGFR_pathway

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PRISM_SPRY4_LQHPLTILPIDQVK_2016-05-05_18-48-57.sky.zip (3 MB)2016-05-06112816
PRISM_SOS1_EYIQPVQLR_2016-05-05_18-46-59.sky.zip (3 MB)2016-05-06112616
PRISM_PTPRE_TGTFIALSNILER_2016-05-05_18-45-53.sky.zip (3 MB)2016-05-06112816
PRISM_GAB1_LTGDPDVLEYYK_2016-05-05_18-45-21.sky.zip (3 MB)2016-05-061121016
PRISM_DUSP6_DSTNLDVLEEFGIK_2016-05-05_18-43-54.sky.zip (3 MB)2016-05-06112816
PRISM_DUSP4_GSVSLEQILPAEEEVR_2016-05-05_18-43-19.sky.zip (3 MB)2016-05-06112816
PRISM_ARAF_TQHCDPEHFPFPAPANAPLQR_2016-05-05_18-07-36.sky.zip (2 MB)2016-05-061121014
PRISM_SPRED1_FGLTFQSPADAR_2016-05-05_18-48-06.sky.zip (3 MB)2016-05-06112816
PRISM_SOS2_LPGYSSAEYR_2016-05-05_18-47-31.sky.zip (3 MB)2016-05-06112816
PRISM_SHOC2_LDSLTTLYLR_2016-05-05_18-46-27.sky.zip (2 MB)2016-05-06112814
PRISM_ERRFl1_NSPSLFPCAPLCER_2016-05-05_18-44-48.sky.zip (3 MB)2016-05-06112816
PRISM_EGFR_LTQLGTFEDHFLSLQR_2016-05-05_18-38-03.sky.zip (2 MB)2016-05-061121012
EGFR_scheduled_SKBR3_2016-05-05_17-44-12.sky.zip (5 MB)2016-05-06326914049712
EGFR_scheduled_NHDF_2016-05-05_17-43-38.sky.zip (5 MB)2016-05-06326914049712
EGFR_scheduled_MCF101_2016-05-05_17-42-47.sky.zip (5 MB)2016-05-06326914049512
EGFR_scheduled_HS578T_2016-05-05_17-41-38.sky.zip (10 MB)2016-05-06316813639618
EGFR_scheduled_HMEC_2016-05-05_17-40-15.sky.zip (5 MB)2016-05-06326914049712
EGFR_scheduled_MD_MBA_2016-05-05_17-43-13.sky.zip (5 MB)2016-05-06326914049513
EGFR_scheduled_MCF7_2016-05-05_17-42-17.sky.zip (6 MB)2016-05-06326914049714
EGFR_scheduled_BT20_2016-05-05_17-38-28.sky.zip (6 MB)2016-05-06326914049716
Conserved protein abundance pattern of the EGFR-MAPK pathway reveals its regulatory architecture

  • Organism: Pacific Northwest National Laboratory
  • Instrument: TSQ Vantage
  • SpikeIn: Yes
Abstract
It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGFR-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundance of most core pathway proteins was between 50,000-70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels(2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.
Experiment Description
To detect pathway proteins by an SRM assay, 10 tryptic peptides without miscleavage(except those peptides containing inhibitory motifs for trypsin) were initially chosen for representing each target protein based upon existing LC-MS/MS results from our own laboratory and public data repositories such as PeptideAtlas, GPM, and PRIDE. For the core pathway proteins without existing LC-MS/MS data, in silico digestion was performed for peptide selection. All selected peptides were unique to the given proteins with no predicted posttranslational modifications. The selected peptides were further evaluated by two prediction tools, the ESP predictor and CONSeQuence software. 5 peptides per protein with moderate hydrophobicity, high spectra counts, and high score from the prediction tools were selected for peptide synthesis. The synthesized crude heavy-isotope labeled peptides were further evaluated for peptide response and fragmentation pattern. For each peptide, 3 transitions were selected based on their abundances and optimal collision energy (CE) values were achieved by direct infusion of the individual peptides and/or multiple LC-SRM runs with CE ramping. Two best response peptides were selected to configure final SRM assays for each target protein, and the best transition (i.e., the one with the most intense SRM signal and without clear evidence of coeluting interference) was used to quantify the target protein. The potential interference for given transitions was assessed based on the relative intensity ratios between the 3 transitions for both light and heavy peptides using an approach similar as previously reported.
Sample Description
Cell pellets from 8 different cell lines were lysed in 100μL of lysis buffer containing 8M urea in 100mM NH4HCO3 pH 7.8. Proteins were reduced by 5mM DTT for 1h at 37°C and alkylated using 20mM iodoacetamide for 1h at room temperature in the dark. Samples were diluted 8-fold with 50mM NH4HCO3 and digested by sequencing grade modified trypsin at a 1:50 enzyme-to-protein ratio (w/w) at 37°C for 3h. Each sample was then desalted by C18 SPE and concentrated to a volume of ~50 μL. The final peptide concentration was measured using BCA assay.
Created on 5/6/16, 11:39 AM