Systems pharmacology using mass spectrometry identifies critical response nodes in prostate cancer
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
H. Alexander Ebhardt
PROVIDER: MSV000081956 | MassIVE | Mon Jan 22 06:22:00 GMT 2018
REPOSITORIES: MassIVE
Publications
Systems pharmacology using mass spectrometry identifies critical response nodes in prostate cancer.
Ebhardt H Alexander HA Root Alex A Liu Yansheng Y Gauthier Nicholas Paul NP Sander Chris C Aebersold Ruedi R
NPJ systems biology and applications 20180702
In the United States alone one in five newly diagnosed cancers in men are prostate carcinomas (PCa). Androgen receptor (AR) status and the PI3K-AKT-mTOR signal transduction pathway are critical in PCa. After initial response to single drugs targeting these pathways resistance often emerges, indicating the need for combination therapy. Here, we address the question of efficacy of drug combinations and development of resistance mechanisms to targeted therapy by a systems pharmacology approach. We ...[more]
Similar Datasets
Project description:Cervicovaginal lavage (CVL)supernatants were heat inactivated for 30 minutes at 56°C before further processing. Total protein concentrations were determined using the Pierce Coomassie Plus (Bradford) Protein Assay (Thermo Scientific, Rockford, IL, USA). Sample protein content and volume were normalised with 25mM ammonium bicarbonate (ABC). Soluble proteins were precipitated using an equal volume of ice cold 30% (w/v) TCA in acetone and incubated at -20⁰C for 2 hours. Samples were centrifuged at 12,000g for 10 minutes (4⁰C) to pellet proteins. Pellets were washed three times with ice cold acetone and allowed to air dry. Further sample processing was performed as previously described (Armstrong et al, 2014) with minor modifications. Briefly, protein pellets were resuspended in 25 mM ABC, 0.05%(w/v) rapigest (Waters), reduced and alkylated. Digestion was performed with proteomic-grade trypsin (Sigma-Aldrich, St. Louis, MO, USA) at a protein:trypsin ratio of 50:1. Rapigest was precipitated by addition of trifluoroacetic acid to a final concentration of 0.5% (v/v). Peptide mixtures were analyzed by on-line nanoflow liquid chromatography using the nanoACQUITY-nLC system (Waters MS technologies) coupled to an LTQ-Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) mass spectrometer equipped with the manufacturer’s nanospray ion source. The gradient of the analytic column (nanoACQUITY UPLCTM BEH130 C18 15cm x 75µm, 1.7µm capillary column) consisted of 3-40% acetonitrile in 0.1% formic acid for 90 min then a ramp of 40-85% acetonitrile in 0.1% formic acid for 5min.
2016-03-15 | PXD003176 | Pride
Project description:In an effort to begin to delineate the bicarbonate regulon, we used microarray analysis with cells grown to late exponential growth phase (3 hr) and then submitted to a 15 min exposure with 0.1 M NaHCO3. Our goal was to define the first set of genes affected by the presence of bicarbonate.
2010-01-06 | E-MEXP-2518 | biostudies-arrayexpress
Project description: Not available
| S-EPMC6026592 | biostudies-literature
Project description:LC-MS/MS analysis of peptide digests of myofibrils from wild type and Dmlc2(Delta 2-46; S66A, S67A) mutant Drosophila flies. Myofibrils and thick filament samples were solubilized in 150 ul 0.1% RapiGest SF Surfactant (Waters Corporation; 50 C, 1 hour). Proteins were reduced by addition of 0.75 ul of 1M dithiothreitol and heating (100 C, 10 min). Cysteines were alkylated by addition of 22.5 ul of 100 mM iodoacetamide in 50 mM ammonium bicarbonate and incubation in the dark (22C, 30 min). Proteins were digested to peptides by addition of 25 ul of 50 mM ammonium bicarbonate containing 5 ug of trypsin (Promega) and incubating (37 C, 18 hours). The samples were dried down in speed vacuum device. Trypsin was deactivated and RapiGest was cleaved by addition of 100 ul of 7% formic acid in 50 mM ammonium bicarbonate and heating (37 C, 1 hour). The resultant peptides were dried down. RapiGest was cleaved again by addition of 100 ul of 0.1% trifluoroacetic acid and heating (37 C, 1 hour). The resultant peptides were dried down and reconstituted a final time in 60 ul of 0.1% trifluoroacetic acid. The samples were centrifuged at 18,800 RCF for 5 minutes (Thermo, Sorvall Legend Micro 21R) to pellet the surfactant. The top 57 ul of solution was transferred into a mass spectrometry (MS) analysis vial.
A 20 ul aliquot of each sample was injected onto an Acquity UPLC HSS T3 column (100 A, 1.8 um, 1 x 150 mm) (Waters Corporation) attached to a UltiMate 3000 ultra-high pressure liquid chromatography (UHPLC) system (Dionex). The peptides were separated and the UHPLC effluent was directly infused into a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (MS) through an electrospray ionization source (Thermo Fisher Scientific). Data were collected in data-dependent MS/MS mode with the top five most abundant ions being selected for fragmentation.
2020-06-24 | MSV000085627 | MassIVE
Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 30 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH 7.5), 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, and proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with trypsin at 37ºC overnight. The resultant peptide solutions were captured on MagCapture HP Tamavidin 2-REV magnetic beads in the presence of 1 mg/ml Pefabloc SC during 3 h of incubation at 4ºC. After washing with TBS five times, the biotinylated peptides were eluted with 100 µL of 1 mM biotin at 95ºC twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrato, and redissolved in 0.1% TFA and 3% ACN.
2020-06-17 | PXD018802 | JPOST Repository
Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 100 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl (pH 8.0) and 2 mM DTT. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, followed by reduction in 5 mM DTT at room temperature for 30 min and alkylation in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with 20 µg trypsin at 37ºC overnight. The resultant peptide solutions were captured on the anti-biotin antibody-coupled beads in the presence of 1 mg/ml Pefabloc SC during 16 h of incubation at 4ºC. After washing with TBS four times, and with ultrapure water twice, the biotinylated peptides were eluted with 50 µL of 0.1% TFA in 80% acetonitrile three times. The combined eluates were evaporated, and desalted using GL-Tip SDB. The desalted eluates were further evaporated, and redissolved in 0.1% TFA.
2021-01-10 | PXD017044 | JPOST Repository
Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 30 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH 7.5), 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, and proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with trypsin at 37ºC overnight. The resultant peptide solutions were captured on anti-biotin (A7C2A) rabbit monoclonal antibody-conjugated beads in the presence of 1 mg/ml Pefabloc SC during 3 h of incubation at 4ºC. After washing with TBS four times, and with ultrapure water twice, the biotinylated peptides were eluted with 100 µL of 0.2% TFA and 80% ACN twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% ACN.
2020-06-17 | PXD018803 | JPOST Repository
Project description:This model is based on paper:
Combination of singularly perturbed vector field method and method of directly defining the inverse mapping applied to complex ODE system prostate cancer model
Author:
Ophir Nave, Miriam Elbaz
Abstract:
We propose a new method to solve a system of complex ordinary differential equations (ODEs) with hidden hierarchy. Given a complex system of the ODE, the hierarchy of the system is generally hidden. Once we reveal the hierarchy of the system, the system can be reduced into subsystems called slow and fast subsystems. This division of slow and fast subsystems reduces the analysis and hence reduces the computation time, which can be expensive. In our new method, we first apply the singularly perturbed vector field method that is the global quasi-linearization method. This method exposes the hierarchy of a given complex system. Subsequently, we apply a version of the homotopy analysis method called the method of directly defining the inverse mapping. We applied our new method to the immunotherapy of advanced prostate cancer.
2019-10-03 | MODEL1910030001 | BioModels
Project description:Sperm isolated from WT mice and PITHD1-deficient mice in triplicate were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl, pH 8.0, and 2 mM DTT. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 × g for 15 min at 4 °C. The supernatants containing 100 µg protein each were reduced in 5 mM DTT at room temperature for 30 min and alkylated in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins were purified by methanol/chloroform precipitation and solubilized with 25 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate buffer. After repeated sonication and vortexing, the proteins were digested with 1 μg of trypsin/Lys-C mix (Promega) for 16 h at 37 °C. Approximately 25 µg of peptides for each sample was labeled with 0.2 mg of TMT6plex reagents for 1 h at room temperature. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with trifluoroacetic acid (TFA), and fractionated using a Pierce High pH Reversed-phase Peptide Fractionation Kit (Thermo Fisher). Nine fractions were collected using 5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 50% acetonitrile (ACN) in 0.1% triethylamine. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA.
2020-01-10 | PXD015347 | JPOST Repository
Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 100 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl (pH 8.0) and 2 mM DTT. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, followed by reduction in 5 mM DTT at room temperature for 30 min and alkylation in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with 20 µg trypsin at 37ºC overnight. The resultant peptide solutions were captured on MagCapture Tamavidin 2-REV magnetic beads in the presence of 1 mg/ml Pefabloc SC during 16 h of incubation at 4ºC. After washing with TBS five times, the biotinylated peptides were eluted with 100 µL of 2 mM biotin at 95ºC twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrato, and redissolved in 0.1% TFA.
2023-04-17 | PXD017042 | JPOST Repository