LINCS PCCSE P100 2020

Phospho-proteomic Profiling Dataset of Chemical Perturbations in Multiple Biological Backgrounds

  • Organism: Homo sapiens
  • Instrument: Q Exactive HF
  • SpikeIn: Yes
  • Keywords: Data-Independent Acquisition, mass spectrometry, proteomics, phospho-signaling, phosphoproteomics
  • Lab head: Malvina Papanastasiou
Abstract
The Library of Integrated Network-based Cellular Signatures (LINCS) Program aims to create a network-based understanding of biology by cataloging changes in gene expression and other cellular processes that occur when cells are exposed to a variety of perturbing agents. The goal of this project is to test the hypothesis that modulation of phosphorylation-mediated signaling events in response to perturbations can establish new cellular states by altering their epigenetic landscape. We have performed mass spectrometry (MS)-based proteomic assays that target quantitative readouts of phospho-signaling and chromatin modifications in cellular models, following perturbation by compounds with varying mechanisms of action. We have employed 128 compounds and treated 5 cancer and 2 neuronal cell lines. The resulting data collected and tools created have been contributed to the LINCS program for the purpose of making connections among disparate perturbations through phosphoproteomics and chromatin modification signatures in concert with other available data types. The data is publicly available on PanoramaPublic and can be queried using Clue (Broad Institute). Analysis of our data will inform novel therapeutic opportunities and synergies, as dysregulation of phospho-signaling and epigenetic systems are two of the most common molecular etiologies identified in a growing number of diseases.
Experiment Description
Detailed P100 protocols can be found in Abelin et al., 2016, MCP and online at https://panoramaweb.org/wiki/LINCS/Overview%20Information/page.view?name=sops. Briefly, cells were lysed after drug treatment with a urea-based buffer that includes phosphatase inhibitors. Proteins (300-500 μg, depending on the cell type) extracted from the cell lysates underwent trypsin digestion followed by desalting using reversed solid phase extraction. Peptides were mixed with a quality-control set of synthetic isotope-labeled peptide standards to monitor the performance of subsequent steps. Phosphopeptide enrichment occurred on an AssayMAP Bravo automated liquid handling platform (Agilent, Santa Clara, CA) using IMAC chemistry. Samples were processed in a 96-well plate format. Prior to mass spectrometry analysis, a second set of synthetic isotope-labelled internal standards for targeted phosphopeptides were spiked into samples to ensure accurate quantification. Data were acquired using a DIA method as described in Abelin et al., 2016, MCP and ion chromatograms were extracted using Skyline (MacLean et al., 2014).
Sample Description
Drug signatures were profiled in various cancer cell lines (A375, YAPC, A549, MCF7, and PC3) and neuronal cell models (Astrocytes & NPCs). Drugs were grouped into four tranches: epigenetically active, neuroactive, kinase inhibitors and cardiotoxic compounds. A detailed description of their mechanism of action can be found online at https://panoramaweb.org/wiki/LINCS/Overview%20Information/page.view?name=LINCS%20PCCSE%20Overview and https://clue.io/touchstone. Drug stocks were dissolved in DMSO for treatment in cell line media and cells were treated in 6 well plates for 3 hours. For cell harvesting, cells from 2-4 wells of a 6 well plate were scraped in lysis buffer and combined per sample. All drug perturbations occurred in triplicates.
Created on 2/7/20, 3:39 PM
PlateLabelCell LineLevel 1Level 2Level 3Level 4Config
Plate67Cardiotoxic DrugsAstrocyte Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate66Kinase InhibitorsAstrocyte Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate65Neuroactive DrugsAstrocyte Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate64Epigenetic DrugsAstrocyte Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate63aCardiotoxic DrugsNPC Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate62Cardiotoxic DrugsYAPC Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate61Cardiotoxic DrugsPC3 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate60Cardiotoxic DrugsMCF7 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate59aCardiotoxic DrugsA375 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate58Cardiotoxic DrugsA549 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate34Kinase InhibitorsPC3 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate33Kinase InhibitorsA549 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate32Kinase Inhibitors (PRM)YAPC Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate31Neuroactive Drugs (PRM)YAPC Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate30Epigenetic DrugsYAPC Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate29_03HKinase Inhibitors (PRM)MCF7 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate28Kinase Inhibitors (PRM)A375 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate27Kinase InhibitorsNPC Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate25Neuroactive Drugs (PRM)A549 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate24Neuroactive DrugsPC3 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate23Neuroactive DrugsMCF7 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate22Neuroactive DrugsA375 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate20Neuroactive DrugsNPC Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate19Epigenetic DrugsA549 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate18Epigenetic Drugs (PRM)PC3 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate17Epigenetic DrugsA375 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate16Epigenetic DrugsNPC Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Plate15Epigenetic DrugsMCF7 Skyline  GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   GCT  [ View in Morpheus  ]   CFG 
Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicatesCalibration Curves
LINCS_P100_DIA_Plate63a_annotated_minimized_2019-11-14_15-28-26.sky.zip2020-02-0790961922,7649496
LINCS_P100_DIA_Plate34_annotated_minimized_2019-08-30_17-25-21.sky.zip2020-02-0790961927529696
LINCS_P100_PRM_Plate28_annotated_minimized_2018-05-02_18-51-02.sky.zip2020-02-0790961921,158960
LINCS_P100_PRM_Plate25_annotated_minimized_2018-05-02_18-24-10.sky.zip2020-02-0790961921,242960
LINCS_P100_DIA_Plate61_annotated_minimized_2018-02-06_17-09-06.sky.zip2020-02-0790961929909596
LINCS_P100_DIA_Plate59a_annotated_minimized_2018-11-30_14-13-58.sky.zip2020-02-0790961922,7649496
LINCS_P100_DIA_Plate33_annotated_minimized_2018-05-02_17-47-07.sky.zip2020-02-0790961927969696
LINCS_P100_DIA_Plate23_annotated_minimized_2018-05-02_18-13-55.sky.zip2020-02-079096192960960
LINCS_P100_DIA_Plate20_annotated_minimized_2018-05-02_21-04-33.sky.zip2020-02-0790961921,0749696
LINCS_P100_DIA_Plate65_annotated_minimized_2019-10-21_16-54-32.sky.zip2020-02-0790961921,5869496
LINCS_P100_DIA_Plate22_annotated_minimized_2018-05-02_22-33-32.sky.zip2020-02-0790961921,0969596
LINCS_P100_DIA_Plate19_annotated_minimized_2018-05-02_17-18-22.sky.zip2020-02-0790961921,1609696
LINCS_P100_DIA_Plate30_annotated_minimized_2018-05-02_18-33-32.sky.zip2020-02-0790961927859696
LINCS_P100_PRM_Plate32_annotated_minimized_2018-05-02_20-45-26.sky.zip2020-02-0790961921,146960
LINCS_P100_DIA_Plate67_annotated_minimized_2018-08-02_13-56-24.sky.zip2020-02-0790961922,7549696
LINCS_P100_DIA_Plate66_annotated_minimized_2018-08-31_10-36-13.sky.zip2020-02-0790961922,7649696
LINCS_P100_PRM_Plate31_annotated_minimized_2018-05-02_18-00-17.sky.zip2020-02-079096192996840
LINCS_P100_PRM_Plate29_03H_annotated_minimized_2018-05-02_21-16-28.sky.zip2020-02-0790961921,5369696
LINCS_P100_DIA_Plate60_annotated_minimized_2017-11-29_16-22-26.sky.zip2020-02-0790961929789596
LINCS_P100_DIA_Plate58_annotated_minimized_2017-11-28_11-50-08.sky.zip2020-02-0790961928349596
LINCS_P100_DIA_Plate24_annotated_minimized_2018-05-02_19-07-44.sky.zip2020-02-0790961927329696
LINCS_P100_PRM_Plate18_annotated_minimized_2020-01-08_17-11-19.sky.zip2020-02-0790961921,5389696
LINCS_P100_DIA_Plate62_annotated_minimized_2018-04-20_15-55-05.sky.zip2020-02-0790961929869696
LINCS_P100_DIA_Plate64_annotated_minimized_2018-05-02_20-34-19.sky.zip2020-02-0790961929829596
LINCS_P100_DIA_Plate27_annotated_minimized_2018-05-02_19-16-47.sky.zip2020-02-0790961921,0649696
LINCS_P100_DIA_Plate16_annotated_minimized_2018-05-02_19-43-10.sky.zip2020-02-0790961921,1229596
LINCS_P100_DIA_Plate17_annotated_minimized_2018-05-02_17-35-29.sky.zip2020-02-0790961921,0549596
LINCS_P100_DIA_Plate15_annotated_minimized_2018-05-02_19-32-22.sky.zip2020-02-0790961921,2029696

This data is available under the CC BY 4.0 license.